Targeting microbial β-CAs: bridging in silico screening with in vitro validation.

Antimicrobial resistance (AMR) is one of the world's most pressing health problems and requires immediate action from the scientific community. Pseudomonas aeruginosa is a particularly concerning bacterium due to its high level of resistance, especially among hospitalized and immunocompromised individuals. We recently investigated PsCA3, one of three P. aeruginosa β‑carbonic anhydrases (β-CAs), as a potential new pharmacological target for developing innovative antibacterial drugs. In this study, we applied a consensus structure-based virtual screening approach to select the most promising PsCA3 inhibitors from an in-house library of 607 small molecules. Twenty-one diverse compounds were selected and experimentally validated through enzyme inhibition assays, which assessed the ability of all ligands to block PsCA3 activity while sparing human α‑carbonic anhydrases (hCAs). Next, we focused on the benzoxazinone/dihydroquinolinone scaffold, testing fifteen additional analogues and expanding the set of assayed microbial β-CAs. Notably, we identified new, potent ligands based on unexplored scaffolds that can effectively target microbial β-CAs at micromolar/submicromolar concentrations with remarkable selectivity over human CAs.