biostudies-literatureChain CU.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)U.S. Department of Health & Human Services | NIH | National Cancer Institute1207-1219https://www.ebi.ac.uk/biostudies/studies/S-EPMC11087268biostudies-literatureUnknown9(5)Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics andNature microbiologyA folate inhibitor exploits metabolic differences in Pseudomonas aeruginosa for narrow-spectrum targeting.PMC11087268NCI-CCSG P30CA072720-5921Rabinowitz JDGitai ZChain CGodbole AKim HGhaffari SSheehan JPXu XFreundlich JSfalseA folate inhibitor exploits metabolic differences in Pseudomonas aeruginosa for narrow-spectrum targeting.Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics and2024-01-01T00:00:00Z2024 May2026-06-01T10:58:30.445Z2025-02-19T00:16:51.676ZS-EPMC110872683859431110.1038/s41564-024-01665-2