Project Summary/Abstract The overuse and misuse of antibiotics has put evolutionary pressure on bacteria to alter or bypass the targets of drugs or otherwise develop resistance, rendering a large percentage of our available medicines and pesticides ineffective. Novel antibiotics have afforded temporary relief due to quick development of resistance, although several pharmaceutical companies have withdrawn from this area of research. Bacterial biofilms further complicate treatment of many bacterial infections. These cell conglomerates contribute to a variety of health conditions and are known to colonize the surfaces of most medical devices. Moreover, they shelter high numbers of persister cells— “dormant” cells which are non-growing and tolerant of most antibiotics. Unfortunately, most existing therapies target metabolic processes which are suspended in these transient subpopulations of bacteria. Altogether we are facing a perfect storm of resistance and tolerance which threatens to kill millions and unravel our current approach to medicine in the process, unless we find a radical solution. To this end, we have identified a potential antibiotic target—the bacterial SOS response. This response to genotoxic stress is conserved across bacteria and has been connected to resistance and tolerance mechanisms, including horizontal gene transfer, mutagenesis, and cell division arrest. Transcription of SOS genes is suppressed by the repressor-protease LexA, which cleaves upon interaction with filamentous protein RecA* to expose the SOS promoter region. A previous high throughput screen identified a potent inhibitor of LexA cleavage. We propose a study to improve this inhibitor and better understand its action and effects. Using a preliminary structure-activity relationship (SAR) study as a guide, we have designed a library of 22-25 analogs for a more in-depth SAR campaign, including analogs specifically designed to overcome potential efflux challenges. Additionally, we have proposed peptide fragments with covalent traps to mimic the native substrate of the LexA protease and irreversibly inhibit its function. Using our most potent inhibitors, we will investigate the downstream biological effects of LexA inhibition, including acquired antibiotic resistance and biofilm formation. We also plan to use photoaffinity probes to identify the inhibitor binding site and orientation within the protein. The uniquely interdisciplinary approach of this proposal will elucidate the mechanism of these inhibitors and will lay the groundwork for a novel strategy to address the resistance and tolerance crisis.