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

Abstract

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 μM. Using isothermal titration calorimetry, a K d of 10 nM for the PqsR ligand binding domain (PqsRLBD) was determined for 61. Furthermore, the crystal structure of 61 with PqsRLBD was attained with a resolution of 2.65 Å. 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.

In vitro Activity of Antivirulence Drugs Targeting the las or pqs Quorum Sensing Against Cystic Fibrosis Pseudomonas aeruginosa Isolates

“…Until now, very few studies showed the effect of QS inhibitors on CF clinical isolates ( Rampioni et al, 2017b ; D’Angelo et al, 2018 ; Baldelli et al, 2020 ; Mahan et al, 2020 ; Papa et al, 2021 ; Soukarieh et al, 2021 ), and only in one study the antivirulence activity of a drug targeting the las QS system, furanone C-30, was tested in a collection of fifty CF isolates, revealing highly variable response and high frequency of resistance to the QS inhibitor in CF isolates ( García-Contreras et al, 2015 ), in overall accordance with this study.…”

“…Nevertheless, since the las QS system is hierarchically dominant over the rhl and pqs system in P. aeruginosa reference strains, the research of QS inhibitors has been targeted mainly toward this system ( Rampioni et al, 2014 ; Soukarieh et al, 2018a ). However, several studies reported the identification of molecules targeting the pqs system, including: metilantranilate ( Calfee et al, 2001 ); farnesol ( Cugini et al, 2007 ); halogenated anthranilic acid ( Lesic et al, 2007 ); quinazolidine derivatives ( Ilangovan et al, 2013 ; Soukarieh et al, 2018b , 2021 ); benzamide-benzimidazole ( Starkey et al, 2014 ; Maura and Rahme, 2017 ); pimozide ( Mellini et al, 2019 ); nitrofurazone and erythromycin estolate ( Baldelli et al, 2020 ); 2-sufonylpyrimidines ( Thomann et al, 2016a , b ). Compared with these anti- pqs drugs, clofoctol has the advantage of being already used in humans for the treatment of pulmonary infections, hence the delivery of this drug to the CF clinical setting should in principle be more straightforward.…”

Investigation of Direct and Retro Chromone-2-Carboxamides Based Analogs of Pseudomonas aeruginosa Quorum Sensing Signal as New Anti-Biofilm Agents

“…Docking of PQS gave a predicted binding energy (E bind ) of −8.1 kcal•mol −1 for the best pose, in which PQS is deeply bonded in the B pocket (Figure 3B), through hydrophobic interactions with the residues Ala102, Ile149, Ala168, and Pro238, contrary to NHQ which occupies the two pockets (Figure 3A). As expected, PQS interactions were mainly hydrophobic [25,29,30]. Our docking results showed PQS forms several H-bonds between its carbonyl group and Gln194 and Ser196, and between its 3-hydroxyl group and Leu197.…”

“…Kinetic studies using pqsR mutants proved that the pqs system is involved in early steps of biofilm formation and supported the fact that it represents an interesting target for new anti-virulence and antibiofilm compounds against P. aeruginosa pathogenicity [27,29,30,48,49]. Regarding the crucial role of the pqs system in the adaptability of P. aeruginosa, including biofilm formation, virulence regulation and some remarkable secondary effects, like iron acquisition or cytotoxicity [26,50,51], we chose an anti-biofilm assay based on detection of adherent cell population, rather than the classically used crystal violet method, suitable for the evaluation of total biomass but nonspecific enough [52].…”

Quorum Sensing Inhibitors to Quench P. aeruginosa Pathogenicity

“…Docking studies suggested that an interaction via a hydrogen bond is possible between the carbonyl function at position 4 of the quinazolone ring and the hydroxyl group of Thr265 as for the 2-alkyl-quinazolone derivative 31. In 2021, Grossman's group described a new quinazolone derivative as a PqsR antagonist (IC 50 of 1.1 µM in a P. aeruginosa PAO1-L strain) with an anti-pyocyanin activity similar to compounds 32 and 33 (80% of inhibition at 3 µM in a P. aeruginosa PAO1-L strain) [111].…”