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

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

Automated summary

This study identified and optimized PqsR antagonists using virtual screening and whole cell assay validation, with compound 61 showing potential as a potent PqsR inhibitor. Compound 61 reduced virulence traits in various strains of P. aeruginosa and enhanced the effect of ciprofloxacin in treating biofilms and infections in Galleria mellonella. These findings suggest compound 61 as a promising lead for developing P. aeruginosa quorum sensing inhibitors for preclinical development.