Project Abstract: Vancomycin-resistant enterococci (VRE) is the second leading cause of drug-resistant hospital-acquired infections (HAIs) in the US, triggering the Centers for Disease Control and Prevention to classify VRE as a serious healthcare threat. Despite the prevalence and severity of VRE infections, there are limited number of effective therapeutic options available for treatment. Moreover, the drugs that are available are also detrimental to the normal gut microbiota ultimately contributing the problematic cycle of microbial imbalance known as dysbiosis, which enterococcus takes advantage of in the first place. Thus, there is a significant need for a treatment that can be used to clear both symptomatic and asymptomatic enterococcus colonization without damaging the homeostasis of normal gut flora. Unlike the costly and time-consuming process of de novo drug discovery, drug repurposing is a novel method to reduce the time, cost and risk associated with drug innovation. Studies proposed in this application build upon discoveries of the potent and narrow spectrum antimicrobial activity of the FDA-approved drugs, carbonic anhydrase inhibitors (CAIs) (acetazolamide, dorzolamide, brinzolamide, ethoxzolamide, methazolamide, and dichlorphenamide), in an applicable clinical range, against highly multidrug-resistant enterococci, including VRE. We have demonstrated both in vitro and in vivo that CAIs are superior to drug of choice, linezolid, and can be used for treatment of serious VRE infections as well as VRE decolonization without harming gut microbiota. In addition, we were able to identify a novel antimicrobial target specific for enterococci that could be exploited in future screening campaigns for new inhibitory scaffolds. Our team has embarked upon medicinal chemistry optimization and improved the potency of the scaffold versus VRE by 570-fold (MIC = 0.007 µg/ml for most potent analog) while maintaining no antibacterial activity against normal gut microbiota. We propose to continue lead optimization and assess the in vivo efficacy in various VRE mouse models as well evaluate the novel inhibitor’s safety and PK profiles to support future lead selection and investigational new drug enabling studies.