Project Summary C. difficile infection (CDI) is triggered by use of broad-spectrum antibiotics which disrupts the natural microbial flora of the gut and allows the Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of the disease requires new strategies for optimal treatment. Furthermore, decreased response rates to metronidazole, high recurrence rates with the use multiple antiboitics, andemergence of multiple antibiotic resistant bacteria demonstrate the urgent need to develop new therapies. A paradigm shift in the general treatment of infectious diseases, including CDI, is necessary to prevent an increase in antibiotic resistance and alternatives to antibiotics should be considered. The goal of this project is to develop a novel non-antibiotic therapy against C. difficile based on the inhibition of the major bacterial virulence factors that mediate the disease. These toxins are post-translationally activated inside intestinal epithelial cells via allosteric activation of their cysteine protease domain (CPD) by the eukaryote-specific small molecule inositol-hexakisphosphate (InsP6). We have developed an activity-based probe that can report on toxin CPD activation. Using this probe, we conducted a high-throughput fluorescence polarization screen to identify novel small-molecule inhibitors of the toxins. The screen revealed a significant number of novel lead compounds as well as an existing phase II clinical drug with nanomolar activity against the toxin. We have used the clinical drug to demonstrate the therapeutic value of blocking toxin function, using both toxigenic and infection models in mice. These studies have confirmed that inhibition of the CPD blocks pathogen-mediated toxicity in host tissues. However, its poor pharmacological properties coupled with its broad target selectivity makes it less than ideal for clinical development for CDI. Therefore, this project will focus in the first 3 years of the project to identify viable lead compounds with improved potency, selectivity, solubility and cellular uptake. We will focus on the clinical lead compound as well as several novel chemical entities (NCEs) identified in our HTS efforts. We will identify three chemically distinct lead molecules and then perform validation in mouse and hamster models of CDI. We will advance one of these molecules (keeping the others as backups) into formulation studies, with the end goal being to identify a single lead molecule and formulation strategy that can move into IND enabling studies after completion of this project.