<b>Optimization of the Acetazolamide Scaffold to Develop Effective Antibiotics against Vancomycin-resistant </b><b><i>Enterococci</i></b>

<p dir="ltr">Vancomycin-resistant <i>Enterococci</i> (VRE) has remained a high-priority bacterial pathogen for the past eight years according to the World Health Organization and is designated a “Serious Threat” by the Center for Disease Control and Prevention. This seemingly harmless member of the human gastrointestinal tract microbiota has acquired multi-drug resistance over past decades, making it a formidable opponent in healthcare settings where it causes serious secondary infections in the bloodstream, tissues, or urinary tract through contamination of central lines, wounds, or catheters. With only one primary standard of care treatment available for treatment of bloodstream VRE, there is a need for new safe and effective therapeutics to enter the antibiotic drug discovery pipeline for targeting VRE. Utilizing previous findings that the acetazolamide (AZM) human carbonic anhydrase (CA) inhibitor scaffold was also potent against VRE, we sought to establish a thorough structure-activity relationship for AZM and optimize potent leads to achieve successful <i>in vivo</i> efficacy in VRE infection models. Based on experimental evidence and literature reports regarding bacterial CAs, our hypothesis is that the AZM scaffold is targeting <i>Enterococcus </i>carbonic anhydrase enzymes as a novel antibiotic target class. First, we report a large cohort of AZM derivatives from which we identified several leads with promising <i>in vitro</i> properties and one primary lead with translatable efficacy against VRE in a septicemic peritonitis mouse model. Secondly, we report chemoproteomic pulldown studies with biotin-functionalized AZM which identified that α-CA from <i>Enterococcus </i>is the primary <i>E. faecium </i>target protein engaged by the AZM scaffold. This work also confirmed that potent modified AZM derivatives are also on-target against α-CA in <i>Enterococcus</i> and translate to potent efficacy <i>in vivo</i>. Finally, we report the lead optimization of an AZM derivative to achieve the first instances of AZM scaffold selectivity for antimicrobial potency over off-target human CA II (hCAII) inhibition. This selectivity over hCAII was observed to contribute to an improved pharmacokinetic profile through reduced <i>in vivo </i>partitioning into red blood cells where hCAII is prominently expressed, allowing for sustained plasma exposure. Altogether, this work lays the foundation for the advancement of AZM-based anti-VRE therapeutics with a novel mechanism of action and enhanced therapeutic window to further assist in combatting a consistently problematic healthcare-acquired pathogen.</p>