Abstract Clostridium difficile infection (CDI) is the main identifiable cause of antibiotic assoicated diarreah. in the US alone, there are close to 500,000 cases annually causing approximately 25,000 deaths and costs the health care system an estimated $6.3 billion. C. difficile spores do not cause disease but can revert to toxin-producing bacteria (a process called germination) in the microbiota-depleted gut of hospitalized patients. We found that synthetic cholan-24-amides inhibit C. difficile spore germination and, more importantly, protects rodents from CDI without toxic effects. While amide-linked bile salts have many attractive properties, their hydrolysis can release toxic byproducts. To address this liability, we developed heterobicyclic non-hydrolysable bile salts (NHBS) as anti- germinants. Importantly, two analogs protect mice from CDI and are stable to the gut microbiome. This R01 renewal is based on five foundational discoveries obtained during the last funding period: (I) Optimized pipeline for CDI prophylactic development, (ii) synthetic bile salt analogs can be uptaked by the host’s enterohepatic circulation (EHC), (iii) bile salt analogs can be modified by the gut microbiota, Bile salt side chain integrity and hydroxylation state influence anti-germination activity and/or CDI prophylaxis, and (v) NHBS analogs are stable towards the gut microbiome, have anti-germination activity against hypervirulent C. difficile spores, and can protect mice from CDI. Based on the sum of these discoveries, we hypothesize that the anti-germination and CDI prophylaxis activities of new NHBS analogs can be modulated by the combined metabolic action of the EHC and the gut microbiota. To achieve these goal, we will: (1) Synthesize libraries of NHBS analogs, (2) characterize the binding sites of anti-germinant binding proteins, (3) determine in vitro efficacy, permeability, stability, and toxicity of NHBS analogs, (4) assess pharmacokinetic parameters of selected NHBS analogs, and (5) determine the pharmacodynamics of CDI prophylactics on bile salt homeostasis At the start of this project, we will finish characterizing our lead NHBS. This data will provide benchmarks for new analogs. Our medicinal chemistry efforts will be initially guided by germination activity, but subsequent identification of NHBS binding sites could aid this effort. As in vitro and in vivo data is obtained, GO/NO GO criteria will eliminate sub-optimal analogs and inform the rational synthesis of more potent and stable anti-germinants. This iterative process will be continued until at least one optimized CDI prophylactic is obtained. As a secondary goal, we will develop CDI prophylactic dosages that can match the heterogenicity of human CDI risk and variable antibiotic regimes.