Combination Therapy Strategy of Quorum Quenching Enzyme and Quorum Sensing Inhibitor in Suppressing Multiple Quorum Sensing Pathways of P. <i>aeruginosa</i>

Abstract The threat of antibiotic resistant bacteria has called for alternative antimicrobial strategies that would mitigate the increase of classical resistance mechanism. Many bacteria employ quorum sensing (QS) to govern the production of virulence factors and formation of drug-resistant biofilms. Targeting the mechanism of QS has proven to be a functional alternative to conventional antibiotic control of infections. However, the presence of multiple QS systems in individual bacterial species poses a challenge to this approach. Quorum sensing inhibitors (QSI) and quorum quenching enzymes (QQE) have been both investigated for their QS interfering capabilities. Here, we first simulated the combination effect of QQE and QSI in blocking bacterial QS. The effect was next validated by experiments using AiiA as QQE and G1 as QSI on Pseudomonas aeruginosa LasR/I and RhlR/I QS circuits. Combination of QQE and QSI almost completely blocked the P. aeruginosa las and rhl QS systems. Our findings provide a potential chemical biology application strategy for bacterial QS disruption

Itaconimides as Novel Quorum Sensing Inhibitors of Pseudomonas aeroginosa

Pseudomonas aeruginosa is known as an opportunistic pathogen that often causes persistent infections associated with high level of antibiotic-resistance and biofilms formation. Chemical interference with bacterial cell-to-cell communication, termed quorum sensing (QS), has been recognized as an attractive approach to control infections and address the drug resistance problems currently observed worldwide. Instead of imposing direct selective pressure on bacterial growth, the right bioactive compounds can preferentially block QS-based communication and attenuate cascades of bacterial gene expression and production of virulence factors, thus leading to reduced pathogenicity. Herein, we report on the potential of itaconimides as quorum sensing inhibitors (QSI) of P. aeruginosa. An initial hit was discovered in a screening program of an in-house compound collection, and subsequent structure-activity relationship (SAR) studies provided analogs that could reduce expression of central QS-regulated virulence factors (elastase, rhamnolipid, and pyocyanin), and also successfully lead to the eradication of P. aeruginosa biofilms in combination with tobramycin. Further studies on the cytotoxicity of compounds using murine macrophages indicated no toxicity at common working concentrations, thereby pointing to the potential of these small molecules as promising entities for antimicrobial drug development.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio

Itaconimides as Novel Quorum Sensing Inhibitors of Pseudomonas aeruginosa

Pseudomonas aeruginosa is known as an opportunistic pathogen that often causes persistent infections associated with high level of antibiotic-resistance and biofilms formation. Chemical interference with bacterial cell-to-cell communication, termed quorum sensing (QS), has been recognized as an attractive approach to control infections and address the drug resistance problems currently observed worldwide. Instead of imposing direct selective pressure on bacterial growth, the right bioactive compounds can preferentially block QS-based communication and attenuate cascades of bacterial gene expression and production of virulence factors, thus leading to reduced pathogenicity. Herein, we report on the potential of itaconimides as quorum sensing inhibitors (QSI) of P. aeruginosa. An initial hit was discovered in a screening program of an in-house compound collection, and subsequent structure-activity relationship (SAR) studies provided analogs that could reduce expression of central QS-regulated virulence factors (elastase, rhamnolipid, and pyocyanin), and also successfully lead to the eradication of P. aeruginosa biofilms in combination with tobramycin. Further studies on the cytotoxicity of compounds using murine macrophages indicated no toxicity at common working concentrations, thereby pointing to the potential of these small molecules as promising entities for antimicrobial drug development

Chemical biology approaches to identify novel anti-pseudomonas aeruginosa agents

The emergence of multidrug-resistance pathogens has called for alternative therapies to treat bacterial infections. Pseudomonas aeruginosa is an opportunistic pathogen that often causes persistent infections associated with highly antibiotic-resistance and biofilms formation. In this study, various chemical biology approaches were utilized to interfere with bacterial cell-to-cell communication, termed quorum sensing (QS), to address drug resistance problems currently observed worldwide. Many bacteria employ QS to govern the production of virulence factors as well as the formation of biofilms. Instead of targeting components essential for growth and stimulate the development of resistant traits, chemical attenuation of QS could preferentially block QS-regulated cascades of bacterial gene expression, thus leading to attenuation of virulence. In many cases, bacteria employ multiple QS systems, which pose a challenge to develop novel inhibitors. In the first study, two different classes of QS interfering agents, such as quorum sensing inhibitor (QSI) and quorum quenching enzyme (QQE), were studied to suppress multiple pathways of QS of P. aeruginosa. The two QS interfering agents have been studied independently and work in different ways. Using mathematical modeling and biochemical assays, the results from this study suggested a novel approach of utilizing combination therapy of QSI and QQE, and also the importance to develop inhibitors that could suppress all QS systems simultaneously and block pathogenesis. In the second study, a drug repurposing approach was utilized to identify novel functions of FDA-approved drugs as QSI against P. aeruginosa. Given the slow pace of the new antibiotics discovery, repurposing of old drugs has been seen as an attractive strategy to recognize the new function of drugs outside their scope of original medical application, with the advantages of safer toxicity profiles, well-published clinical data, and lower cost and time. Five drugs with excellent QSI activities, namely benzbromarone (uricosuric agent), 8-quinolinol sulfate monohydrate (antineurodegenerative, anticancer, and antidiabetic agent), cetylpyridium chloride (antiseptic agent), zinc pyrithione (antidandruff and antifungal agent), and carbimazole (hyperthyroidism drug) were successfully identified. The application of the compounds in vivo using peritoneal silicone implant models on mice study was also demonstrated. Overall, this study represents how drug repurposing approach could be used to identify repurposable compounds as novel QS-interfering compounds to attenuate the virulence of P. aeruginosa. In the last chapter, a small chemical library based on zinc pyrithione was constructed as part of Structure-Activity Relationship (SAR) study. Novel metal-containing compounds were successfully synthesized and characterized as QSI of P. aeruginosa. SAR study yielded analogs that could reduce the production of QS-regulated virulence factors, as well as successfully eradicate colistin-resistant cells in P. aeruginosa biofilms when used together with the antibiotic. Transcriptomic profiling using RNA sequencing as well as experimental adaptive evolution study reveal the working mechanism of the compounds. As ionophore, the compounds could deliver metal ions through lipid membrane, and once inside the cells, the released metal ions interact with several targets simultaneously and also affect metal homeostasis. Both fur and zur pathways are affected upon treatment with compounds. The findings presented in this study show potentials of chemical attenuation for treating P. aeruginosa infections.Doctor of Philosoph

Disulfide Bond-Containing Ajoene Analogues As Novel Quorum Sensing Inhibitors of Pseudomonas aeruginosa

Since its discovery 22 years ago, the bacterial cell-to-cell communication system, termed quorum sensing (QS), has shown potential as antipathogenic target. Previous studies reported that ajoene from garlic inhibits QS in opportunistic human pathogen Pseudomonas aeruginosa. In this study, screening of an in-house compound library revealed two sulfur-containing compounds which possess structural resemblance with ajoene and inhibit QS in bioreporter assay. Following a quantitative structure–activity relationship (SAR) study, 25 disulfide bond-containing analogues were synthesized and tested for QS inhibition activities. SAR study indicated that the allyl group could be replaced with other substituents, with the most active being benzothiazole derivative (IC₅₀ = 0.56 μM). The compounds were able to reduce QS-regulated virulence factors (elastase, rhamnolipid, and pyocyanin) and successfully inhibit P. aeruginosa infection in murine model of implant-associated infection. Altogether, the QS inhibition activity of the synthesized compounds is encouraging for further exploration of novel analogues in antimicrobial drug development

Discovery, biosynthesis and antifungal mechanism of the polyene-polyol meijiemycin

Produced by a newly isolated Streptomycetes strain, meijiemycin is a gigantic linear polyene-polyol that exhibits structural features not seen in other members of the polyene-polyol family. We propose a biosynthetic mechanism and demonstrate that meijiemycin inhibits hyphal growth by inducing the aggregation of ergosterol and restructuring of the fungal plasma membrane.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Accepted versio

Low-dose anti-inflammatory combinatorial therapy reduced cancer stem cell formation in patient-derived preclinical models for tumour relapse prevention

Background: Emergence of drug-resistant cancer phenotypes is a challenge for anti-cancer therapy. Cancer stem cells are identified as one of the ways by which chemoresistance develops. Method: We investigated the anti-inflammatory combinatorial treatment (DA) of doxorubicin and aspirin using a preclinical microfluidic model on cancer cell lines and patient-derived circulating tumour cell clusters. The model had been previously demonstrated to predict patient overall prognosis. Results: We demonstrated that low-dose aspirin with a sub-optimal dose of doxorubicin for 72 h could generate higher killing efficacy and enhanced apoptosis. Seven days of DA treatment significantly reduced the proportion of cancer stem cells and colony-forming ability. DA treatment delayed the inhibition of interleukin-6 secretion, which is mediated by both COX-dependent and independent pathways. The response of patients varied due to clinical heterogeneity, with 62.5% and 64.7% of samples demonstrating higher killing efficacy or reduction in cancer stem cell (CSC) proportions after DA treatment, respectively. These results highlight the importance of using patient-derived models for drug discovery. Conclusions: This preclinical proof of concept seeks to reduce the onset of CSCs generated post treatment by stressful stimuli. Our study will promote a better understanding of anti-inflammatory treatments for cancer and reduce the risk of relapse in patients.Singapore. National Medical Research Council (Grant NMRC

Low-dose anti-inflammatory combinatorial therapy reduced cancer stem cell formation in patient-derived preclinical models for tumour relapse prevention

10.1038/s41416-018-0301-9BRITISH JOURNAL OF CANCER1204407-42