Design and Evaluation of New Quinazolin-4(3<i>H</i>)-one Derived PqsR Antagonists as Quorum Sensing Quenchers in <i>Pseudomonas aeruginosa</i>

P. aeruginosa (PA) continues to pose a threat global public health due its high levels of antimicrobial resistance (AMR). The ongoing AMR crisis has led an alarming shortage effective treatments for resistant microbes, and hence there is pressing demand the development novel interventions. potential use antivirulence therapeutics tackle bacterial infections attracted considerable attention over past decades as they hamper pathogenicity target microbes with reduced selective pressure, minimizing emergence resistance. One such approach interfere PA pqs quorum sensing system which upon interaction PqsR, Lys-R type transcriptional regulator, cognate signal molecules 4-hydroxy-2-heptylquinoline (HHQ) 2-heptyl-3-hydroxy-4-quinolone (PQS), governs multiple virulence traits host–microbe interactions. In this study, we report hit identification optimization PqsR antagonists using virtual screening coupled whole cell assay validation. optimized compound 61 ((R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)acetonitrile) was found inhibit expression PpqsA promoter controlled by IC50 1 ?M. Using isothermal titration calorimetry, Kd 10 nM ligand binding domain (PqsRLBD) determined 61. Furthermore, crystal structure PqsRLBD attained resolution 2.65 Å. Compound significantly pyocyanin, PQS, HHQ in PAO1-L, PA14 lab strains PAK6085 clinical isolate. potentiated effect ciprofloxacin early stages biofilm treatment Galleria mellonella infected PA. Altogether, data shows potent inhibitor lead toward QS can be advanced into preclinical development.