A Small-Molecule Inhibitor of the Anthranilyl-CoA Synthetase PqsA for the Treatment of Multidrug-Resistant Pseudomonas aeruginosa

One of the challenges associated with the treatment of Pseudomonas aeruginosa infections is the high prevalence of multidrug resistance (MDR). Since conventional antibiotics are ineffective at treating such bacterial infections, innovative antibiotics acting upon novel targets or via mechanisms are urgently required. In this study, we identified a quorum sensing inhibitor (QSI), norharmane, that uniquely shows weak antibacterial activity but strongly inhibits pyocyanin production and biofilm formation of MDR P. aeruginosa. Biophysical experiments and molecular docking studies showed that norharmane competes with anthraniloyl-AMP for anthranilyl-CoA synthetase PqsA of P. aeruginosa at the ligand-binding pocket, which is not exploited by current inhibitors, thereby altering transcription regulatory activity. Moreover, norharmane exhibits synergy with polymyxin B. This synergism exhibits a high killing rate, low probability of resistance selection, and minimal cytotoxicity. Notably, norharmane can effectively boost polymyxin B activity against MDR P. aeruginosa-associated infections in animal models. Together, our findings provide novel insight critical to the design of improved PqsA inhibitors, and an effective combination strategy to overcome multiantibiotic bacterial resistance using conventional antibiotics and QSIs. IMPORTANCE Pseudomonas aeruginosa is a dominant hospital-acquired bacterial pathogen typically found in immunocompromised individuals. It is particularly dangerous for patients with chronic lung diseases and was identified as a serious threat for patients in the 2019 Antimicrobial Resistance Threats report (https://www.cdc.gov/drugresistance/biggest-threats.html). In this study, we used activity-based high-throughput screening to identify norharmane, a potent and selective inhibitor of P. aeruginosa PqsA, which is a well-conserved master quorum sensing (QS) regulator in multidrug resistant (MDR) P. aeruginosa. This compound competitively binds anthranilyl-CoA synthetase PqsA at the anthraniloyl-AMP binding domain, which has not been exploited by known inhibitors. Remarkably, norharmane can significantly block the production of the virulence factor, pyocyanin (87%), and biofilm formation (80%) in MDR P. aeruginosa. Furthermore, norharmane is capable of augmenting polymyxin B activity against MDR P. aeruginosa in cell cultures and animal models. Taken together, these results suggest that norharmane may be an effective adjuvant for combating multiantibiotic bacterial resistance.