Project Abstract: Gonorrhea is a sexually transmitted disease caused by the bacterial pathogen Neisseria gonorrhoeae that colonizes urogenital, anal, and nasopharyngeal tissues. Locally in the United States the Centers for Disease Control and Prevention (CDC) reported a 67% increase of gonorrhea cases between 2013 – 2017 with >550,000 cases in 2017 alone. N. gonorrhoeae wreaks havoc on world health care systems causing pelvic inflammatory disease, infertility and ectopic pregnancies. The bacteria can also be transmitted from mother to child during birth and lead to blindness. If left untreated N. gonorrhoeae can cause gonococcemia resulting in skin infection, arthritis or endocarditis. Pathogenic gonorrhea strains are increasingly resistant to common front-line antibiotics. The WHO surveillance program reports resistance to most available antibiotics. Rampant resistance has caused the CDC and the World Health Organization each to classify N. gonorrhoeae as a superbug and a future with an untreatable gonococcal infection is a real possibility. Thus, there is significant unmet need to identify novel targets and molecules with therapeutic potential. Studies proposed in this application build upon discoveries that FDA- approved carbonic anhydrase inhibitors (CAIs), such as acetazolamide and ethoxzolamide, display potent antimicrobial activity, in an applicable clinical range, against N. gonorrhoeae. CAIs, and analogs we have designed, also have no antimicrobial effect on commensal bacteria reducing the potential for problematic dysbiosis caused by antibiotic treatment. We have shown that the molecules exhibit their antibiotic effect by inhibiting the carbonic anhydrase from N. gonorrhoeae and have validated N. gonorrhoeae carbonic anhydrase (NgCA) as a viable anti-gonococcal therapeutic target. Our team has improved the potency of the CAI-based inhibitors from 4 µg/mL to 0.5 µg/mL. This proposal will continue lead optimization of CAI-based analogs using structure-based design while incorporating modifications to improve permeability into the Gram-negative cell. Molecules will be assessed in in vitro antimicrobial assays and prioritized analogs will progress to in vitro pharmacokinetic (PK) and pharmacologic profiling. Finally, top performing analogs will be assessed for in vivo efficacy in various gonorrhea mouse models as well evaluated the in safety and pharmacokinetic assay to support future lead selection and investigational new drug enabling studies.