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.

Targeting Bacterial Central Metabolism for Drug Development

John McKinney, Paul Murima

Current antibiotics, derived mainly from natural sources, inhibit a narrow spectrum of cellular processes, namely DNA replication, protein synthesis, and cell wall biosynthesis. With the worldwide explosion of drug resistance, there is renewed interest in the investigation of alternate essential cellular processes, including bacterial central metabolic pathways, as a drug target space for the next generation of antibiotics. However, the validation of targets in central metabolism is more complex, as essentiality of such targets can be conditional and/or contextual. Bearing in mind our enhanced understanding of prokaryotic central metabolism, a key question arises: can central metabolism be bacteria's Achilles' heel and a therapeutic target for the development of new classes of antibiotics? In this review, we draw lessons from oncology and attempt to address some of the open questions related to feasibility of targeting bacterial central metabolism as a strategy for developing new antibacterial drugs.

Elsevier2014

OprD Repression upon Metal Treatment Requires the RNA Chaperone Hfq in Pseudomonas aeruginosa

Rodrigo Manuel Gonzalez, Tiziana Scrignari

The metal-specific CzcRS two-component system in Pseudomonas aeruginosa is involved in the repression of the OprD porin, causing in turn carbapenem antibiotic resistance in the presence of high zinc concentration. It has also been shown that CzcR is able to directly regulate the expression of multiple genes including virulence factors. CzcR is therefore an important regulator connecting (i) metal response, (ii) pathogenicity and (iii) antibiotic resistance in P. aeruginosa. Recent data have suggested that other regulators could negatively control oprD expression in the presence of zinc. Here we show that the RNA chaperone Hfq is a key factor acting independently of CzcR for the repression of oprD upon Zn treatment. Additionally, we found that an Hfq-dependent mechanism is necessary for the localization of CzcR to the oprD promoter, mediating oprD transcriptional repression. Furthermore, in the presence of Cu, CopR, the transcriptional regulator of the CopRS two-component system also requires Hfq for oprD repression. Altogether, these results suggest important roles for this RNA chaperone in the context of environment-sensing and antibiotic resistance in P. aeruginosa.

Mdpi Ag2016

Ecological strategies and metabolic trade-offs of complex environmental biofilms

Tom Ian Battin, Hannes Markus Peter

Microorganisms aggregated into matrix-enclosed biofilms dominate microbial life in most natural, engineered, and medical systems. Despite this, the ecological adaptations and metabolic trade-offs of the formation of complex biofilms are currently poorly understood. Here, exploring the dynamics of bacterial ribosomal RNA operon (rrn) copy numbers, we unravel the genomic underpinning of the formation and success of stream biofilms that contain hundreds of bacterial taxa. Experimenting with stream biofilms, we found that nascent biofilms in eutrophic systems had reduced lag phases and higher growth rates, and more taxa with higher rrn copy number than biofilms from oligotrophic systems. Based on these growth-related traits, our findings suggest that biofilm succession was dominated by slow-but-efficient bacteria likely with leaky functions, such as the production of extracellular polymeric substances at the cost of rapid growth. Expanding our experimental findings to biofilms from 140 streams, we found that rrn copy number distribution reflects functional trait allocation and ecological strategies of biofilms to be able to thrive in fluctuating environments. These findings suggest that alternative trade-offs dominating over rate-yield trade-offs contribute to the evolutionary success of stream biofilms.

Nature Publishing Group2017