In silico and in vitro-guided identification of inhibitors of alkylquinolone-dependent quorum sensing in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis, wound and nosocomial infections, posing a serious burden to public health, due to its antibiotic resistance. The P. aeruginosa Pseudomonas Quinolone System (pqs) quorum sensing system, driven by the activation of the transcriptional regulator, PqsR (MvfR) by alkylquinolone (AQ) signal molecules, is a key player in the regulation of virulence and a potential target for the development of novel antibacterial agents. In this study, we performed in silico docking analysis, coupled with screening using a P. aeruginosa mCTX::PpqsA-lux chromosomal promoter fusion, to identify a series of new PqsR antagonists. The hit compounds inhibited pyocyanin and alkylquinolone signal molecule production in P. aeruginosa PAO1-L and PA14 strains. The inhibitor Ia, which showed the highest activity in PA14, reduced biofilmformation in PAO1-L and PA14, increasing their sensitivity to tobramycin. Furthermore, the hepatic and plasma stabilities for these compounds were determined in both rat and human in vitro microsomal assays, to gain a further understanding of their therapeutic potential. This work has uncovered a new class of P. aeruginosa PqsR antagonists with potential for hit to lead optimisation in the search for quorum sensing inhibitors for future anti-infective drug discovery programs

Phylogenetic congruence and ecological coherence in terrestrial Thaumarchaeota

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. Acknowledgements We would like to thank Dr Robert Griffith/CEH for providing DNA from soil samples and Dr Anthony Travis for his help with BioLinux. Sequencing was performed in NERC platform in Liverpool. CG-R was funded by a NERC fellowship NE/J019151/1. CQ was funded by a MRC fellowship (MR/M50161X/1) as part of the cloud infrastructure for microbial genomics consortium (MR/L015080/1).Peer reviewedPublisher PD

Pseudomonas aeruginosa PQS mediated virulence regulation and interference

Pseudomonas aeruginosa is a ubiquitous bacterium that can be found in most mesophilic aquatic and terrestrial habitats. Furthermore, P. aeruginosa (PA) is an important opportunistic pathogen that can infect humans, animals, insects and plants. P. aeruginosa has three hierarchically organised quorum sensing (QS) systems named las, rhl and pqs. The las and rhl are classic QS systems that use an N-acylhomoserine lactone as autoinducers. The las system is the major QS system of the cell and controls the other two and there is partial redundancy with the rhl system regarding the genes and functions controlled by the system. Activated functions are related to motility, virulence and biofilm formation. The pqs system is a QS system based in alkyl-4(1H)-quinolones (AQs) molecules, specifically the 2-heptyl-3,4-dihydroxyquinoline called pseudomonas quinolone factor (PQS). The pqs system is under control of las and rhl and can in turn influence the expression of rhl. The pqs system controls virulence, iron acquisition, biofilm maturation and the oxidative stress response. The PqsR is the main regulator of the pqs system upon activation with PQS. The inhibition of QS and in particular the pqs system is an approach to decrease the virulence of P. aeruginosa in vivo to improve the outcome of antibiotic treatments and decrease the P. aeruginosa associated morbidity. The SENBIOTAR project (sensitising Pseudomonas aeruginosa biofilms to antibiotics and reducing virulence through novel target inhibition, MRC project MR/N501852/1) aims at developing PqsR antagonists that inhibit the pqs system. A series of potential inhibitors were tested for activity in a pqsA-lux transcriptional reporter in Chapter 3. Two compounds, SEN016 and SEN066 were found to actively inhibit expression and SEN016 was used for further in silico optimisation, developing a series of compounds that were tested for IC50 and isothermal titration calorimetry (ITC). The lead compound SEN089 showed a K(D) (dissociation constant) of 2.66 nM and IC50 of 67 nM while compounds SEN022 and SEN066 did not bind to PqsRLBD even if they inhibited pqs. The compounds were tested in a variety of phenotypes and strains. Results from Chapter 4 show that compounds effectively inhibit phenotypes under pqs regulation but not others controlled by different regulatory systems. The data evidenced significant differences on the efficacy of compounds when tested in other strains. Notably, SEN089 remained the overall best compound. Moreover, data provided evidence that P. aeruginosa has at least two autolytic mechanisms, one is activated by pqs whereas the other is repressed by PQS. A novel hypothesis for the role of PQS in the CF lung is also discussed there. In Chapter 3, some compounds were tested in biofilm models to assess the relevance of pqs inhibition. There was a significant increased bioactivity between the compounds and antibiotics tested as well as between the compounds and shearing forces. Several guidelines are provided for optimal confocal imaging aimed at quantitation. Furthermore, another novel hypothesis is presented regarding the role of pqs in the biofilm of the CF lung describing it as a response mechanism rather than an active system. In summary, a series of PqsR antagonists were developed and analysed for binding affinity to PqsR, secondary unspecific activity, pqs inhibition, biofilm impact and an additive effect on antibiotic treatment in different strains of P. aeruginosa. The compounds successfully inhibit pqs and had a significant impact in virulence modulation as well as sensitivity towards tobramycin or ciprofloxacin. This work explores the inhibition of pqs as an effective therapeutic target and suggests multiple novel mechanisms through which pqs regulates the physiology of the cell

Pseudomonas aeruginosa PQS mediated virulence regulation and interference

Pseudomonas aeruginosa is a ubiquitous bacterium that can be found in most mesophilic aquatic and terrestrial habitats. Furthermore, P. aeruginosa (PA) is an important opportunistic pathogen that can infect humans, animals, insects and plants. P. aeruginosa has three hierarchically organised quorum sensing (QS) systems named las, rhl and pqs. The las and rhl are classic QS systems that use an N-acylhomoserine lactone as autoinducers. The las system is the major QS system of the cell and controls the other two and there is partial redundancy with the rhl system regarding the genes and functions controlled by the system. Activated functions are related to motility, virulence and biofilm formation. The pqs system is a QS system based in alkyl-4(1H)-quinolones (AQs) molecules, specifically the 2-heptyl-3,4-dihydroxyquinoline called pseudomonas quinolone factor (PQS). The pqs system is under control of las and rhl and can in turn influence the expression of rhl. The pqs system controls virulence, iron acquisition, biofilm maturation and the oxidative stress response. The PqsR is the main regulator of the pqs system upon activation with PQS. The inhibition of QS and in particular the pqs system is an approach to decrease the virulence of P. aeruginosa in vivo to improve the outcome of antibiotic treatments and decrease the P. aeruginosa associated morbidity. The SENBIOTAR project (sensitising Pseudomonas aeruginosa biofilms to antibiotics and reducing virulence through novel target inhibition, MRC project MR/N501852/1) aims at developing PqsR antagonists that inhibit the pqs system. A series of potential inhibitors were tested for activity in a pqsA-lux transcriptional reporter in Chapter 3. Two compounds, SEN016 and SEN066 were found to actively inhibit expression and SEN016 was used for further in silico optimisation, developing a series of compounds that were tested for IC50 and isothermal titration calorimetry (ITC). The lead compound SEN089 showed a K(D) (dissociation constant) of 2.66 nM and IC50 of 67 nM while compounds SEN022 and SEN066 did not bind to PqsRLBD even if they inhibited pqs. The compounds were tested in a variety of phenotypes and strains. Results from Chapter 4 show that compounds effectively inhibit phenotypes under pqs regulation but not others controlled by different regulatory systems. The data evidenced significant differences on the efficacy of compounds when tested in other strains. Notably, SEN089 remained the overall best compound. Moreover, data provided evidence that P. aeruginosa has at least two autolytic mechanisms, one is activated by pqs whereas the other is repressed by PQS. A novel hypothesis for the role of PQS in the CF lung is also discussed there. In Chapter 3, some compounds were tested in biofilm models to assess the relevance of pqs inhibition. There was a significant increased bioactivity between the compounds and antibiotics tested as well as between the compounds and shearing forces. Several guidelines are provided for optimal confocal imaging aimed at quantitation. Furthermore, another novel hypothesis is presented regarding the role of pqs in the biofilm of the CF lung describing it as a response mechanism rather than an active system. In summary, a series of PqsR antagonists were developed and analysed for binding affinity to PqsR, secondary unspecific activity, pqs inhibition, biofilm impact and an additive effect on antibiotic treatment in different strains of P. aeruginosa. The compounds successfully inhibit pqs and had a significant impact in virulence modulation as well as sensitivity towards tobramycin or ciprofloxacin. This work explores the inhibition of pqs as an effective therapeutic target and suggests multiple novel mechanisms through which pqs regulates the physiology of the cell

BaiJ and BaiB are key enzymes in the chenodeoxycholic acid 7a-dehydroxylation pathway in the gut microbe Clostridium scindens ATCC 35704

Data set for bile acid quantification for submitted manuscript: BaiJ and BaiB are key enzymes in the chenodeoxycholic acid 7a-dehydroxylation pathway in the gut microbe Clostridium scindens ATCC 35704. By Karin Lederballe Meibom et al

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

Hit Identification of New Potent PqsR Antagonists as Inhibitors of Quorum Sensing in Planktonic and Biofilm Grown Pseudomonas aeruginosa

Current treatments for Pseudomonas aeruginosa infections are becoming less effective because of the increasing rates of multi-antibiotic resistance. Pharmacological targeting of virulence through inhibition of quorum sensing (QS) dependent virulence gene regulation has considerable therapeutic potential. In P. aeruginosa, the pqs QS system regulates the production of multiple virulence factors as well as biofilm maturation and is a promising approach for developing antimicrobial adjuvants for combatting drug resistance. In this work, we report the hit optimisation for a series of potent novel inhibitors of PqsR, a key regulator of the pqs system, bearing a 2-((5-methyl-5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio) acetamide scaffold. The initial hit compound 7 (PAO1-L IC50 0.98 +/- 0.02 mu M, PA14 inactive at 10 mu M) was obtained through a virtual screening campaign performed on the PqsR ligand binding domain using the University of Nottingham Managed Chemical Compound Collection. Hit optimisation gave compounds with enhanced potency against strains PAO1-L and PA14, evaluated using P. aeruginosa pqs-based QS bioreporter assays. Compound 40 (PAO1-L IC50 0.25 +/- 0.12 mu M, PA14 IC50 0.34 +/- 0.03 mu M) is one of the most potent PqsR antagonists reported showing significant inhibition of P. aeruginosa pyocyanin production and pqs system signaling in both planktonic cultures and biofilms. The co-crystal structure of 40 with the PqsR ligand binding domain revealed the specific binding interactions occurring between inhibitor and this key regulatory protein

Design, Synthesis and Evaluation of New 1H- benzo[d]imidazole based PqsR Inhibitors as Adjuvant Therapy for Pseudomonas aeruginosa Infections

Pseudomonas aeruginosa is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel 2 antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signalling system controlling virulence, has emerged as a promising approach as an antibiotic adjuvant therapy. Interference with the pqs system, one of three QS systems in P. aeruginosa, results in reduction of bacterial virulence gene expression and biofilm maturation. Herein, we report a hit to lead process to fine tune the potency of our previously reported inhibitor 1 (IC50 3.2 μM in P. aeruginosa PAO1-L) which led to the discovery of 2- (4-(3-((6-chloro-1-isopropyl-1H-benzo[d]imidazol-2-yl)amino)-2-hydroxypropoxy)phenyl)acetonitrile (6f) as a potent PqsR antagonist. Compound 6f inhibited the PqsR-controlled PpqsA-lux transcriptional reporter fusion in P. aeruginosa at low sub-micromolar concentrations. Moreover, 6f showed improved efficacy against P. aeruginosa CF isolates with significant inhibition of pyocyanin, 2-alkyl-4(1H)- quinolones productio