Antibiotic resistance is a growing and increasingly serious public health problem. Infectious diseases caused by Escherichia coli and other bacteria are responsible for millions of deaths each year, and much of this mortality is due to the rise of antibiotic resistant organisms. Because antibiotic-resistant infections double the duration of hospital stay, mortality, and morbidity as compared with drug-susceptible infections, economic costs of antibiotic resistance are estimated to be in the billions of dollars. The overall goal of this project is to develop new anti-infectives that are highly effective and refractory to antibiotic resistance using a Combinatorial Genetic Technology (CGT) that allows the identification of new rRNA target sites and the specific nucleotides that are essential for functionality and viability, and RNA Homology Modeling software that allows accurate prediction of mutant RNA structures. Phase I of this project was highly successful. A functional mutation library of E. coli 16S rRNA was constructed and ~5000 viable clones were sequenced. Using CGT, 67 regions of E. coli rRNA that contain nucleotides essential for viability were identified. The 67 functionally important regions include known binding sites for antibiotics, tRNAs, proteins, the large ribosomal subunit and initiation factors. Also included were a number of sites that are clearly essential for ribosome function, but for which no functional role has been identified to date. Some of the individual regions occur near each other in 30S subunit crystal structures and probably contribute to a single functional role. The Phase II specific aims are 1) to select one RNA subdomain as a prioritized target from the four potential targets chosen from the RNA "regions of interest" identified in Phase I; 2) to use CGT to identify every mutation of the target that could lead to drug resistance, and use multidimensional NMR spectroscopy and homology modeling to determine the essential structural components of the target; 3) to screen compound libraries against the wild type target and its viable mutants; and 4) to carry out structural studies of target/hit complexes to allow optimization of hit compounds, and validate the target/compound using in vitro and in vivo assays of antibacterial activity. RiboNovix will complete the work necessary to develop drug candidates from the leads, and will move qualified candidates into pre-clinical development. Anti-infectives developed against the target identified in this study will likely be highly effective against microbial pathogens and resistant to target site mutation, thus resulting in drugs refractory to antibiotic resistance. Antibiotic resistance is a growing and increasingly serious public health problem. Infectious diseases caused by Escherichia coli and other bacteria are responsible for millions of deaths each year, and much of this mortality is due to the rise of antibiotic resistant organisms. The overall goal of this project is to develop new anti-infectives that are highly effective and refractory to antibiotic resistance. [unreadable] [unreadable] [unreadable]