Synthesis,Antimicrobial, Anti-Virulence and Anticancer Evaluation of New 5(4H)-Oxazolone-Based Sulfonamides

Since the synthesis of prontosil the first prodrug shares their chemical moiety, sulfonamides exhibit diverse modes of actions to serve as antimicrobials, diuretics, antidiabetics, and other clinical applications. This inspiring chemical nucleus has promoted several research groups to investigate the synthesis of new members exploring new clinical applications. In this study, a novel series of 5(4H)-oxazolone-based-sulfonamides (OBS) 9a–k were synthesized, and their antibacterial and antifungal activities were evaluated against a wide range of Gram-positive and -negative bacteria and fungi. Most of the tested compounds exhibited promising antibacterial activity against both Gram-positive and -negative bacteria particularly OBS 9b and 9f. Meanwhile, compound 9h showed the most potent antifungal activity. Moreover, the OBS 9a, 9b, and 9f that inhibited the bacterial growth at the lowest concentrations were subjected to further evaluation for their anti-virulence activities against Pseudomonas aeruginosa and Staphylococcus aureus. Interestingly, the three tested compounds reduced the biofilm formation and diminished the production of virulence factors in both P. aeruginosa and S. aureus. Bacteria use a signaling system, quorum sensing (QS), to regulate their virulence. In this context, in silico study has been conducted to assess the ability of OBS to compete with the QS receptors. The tested OBS showed marked ability to bind and hinder QS receptors, indicating that anti-virulence activities of OBS could be due to blocking QS, the system that controls the bacterial virulence. Furthermore, anticancer activity has been further performed for such derivatives. The OBS compounds showed variable anti-tumor activities, specifically 9a, 9b, 9f and 9k, against different cancer lines. Conclusively, the OBS compounds can serve as antimicrobials, anti-virulence and anti-tumor agents.     

Baicalin Represses Type Three Secretion System of Pseudomonas aeruginosa through PQS System

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against Pseudomonas aeruginosa from Chinese herbs and investigated baicalin from Scutellariae radix as such an active anti-virulence compound. The effect of baicalin on a range of important virulence factors in P. aeruginosa was assessed using luxCDABE-based reporters and by phenotypical assays. The molecular mechanism of the virulence inhibition by baicalin was investigated using genetic approaches. The impact of baicalin on P. aeruginosa pathogenicity was evaluated by both in vitro assays and in vivo animal models. The results show that baicalin diminished a plenty of important virulence factors in P. aeruginosa, including the Type III secretion system (T3SS). Baicalin treatment reduced the cellular toxicity of P. aeruginosa on the mammalian cells and attenuated in vivo pathogenicity in a Drosophila melanogaster infection model. In a rat pulmonary infection model, baicalin significantly reduced the severity of lung pathology and accelerated lung bacterial clearance. The PqsR of the Pseudomonas quinolone signal (PQS) system was found to be required for baicalin’s impact on T3SS. These findings indicate that baicalin is a promising therapeutic candidate for treating P. aeruginosa infections.     

Assessing the Molecular Targets and Mode of Action of Furanone C-30 on Pseudomonas aeruginosa Quorum Sensing

Quorum sensing (QS), a sophisticated system of bacterial communication that depends on population density, is employed by many pathogenic bacteria to regulate virulence. In view of the current reality of antibiotic resistance, it is expected that interfering with QS can address bacterial pathogenicity without stimulating the incidence of resistance. Thus, harnessing QS inhibitors has been considered a promising approach to overriding bacterial infections and combating antibiotic resistance that has become a major threat to public healthcare around the globe. Pseudomonas aeruginosa is one of the most frequent multidrug-resistant bacteria that utilize QS to control virulence. Many natural compounds, including furanones, have demonstrated strong inhibitory effects on several pathogens via blocking or attenuating QS. While the natural furanones show no activity against P. aeruginosa, furanone C-30, a brominated derivative of natural furanone compounds, has been reported to be a potent inhibitor of the QS system of the notorious opportunistic pathogen. In the present study, we assess the molecular targets and mode of action of furanone C-30 on P. aeruginosa QS system. Our results suggest that furanone C-30 binds to LasR at the ligand-binding site but fails to establish interactions with the residues crucial for the protein’s productive conformational changes and folding, thus rendering the protein dysfunctional. We also show that furanone C-30 inhibits RhlR, independent of LasR, suggesting a complex mechanism for the agent beyond what is known to date.     

Short-chain reactive probes as tools to unravel the Pseudomonas aeruginosa quorum sensing regulon

In recent years, the world has seen a troubling increase in antibiotic resistance among bacterial pathogens. In order to provide alternative strategies to combat bacterial infections, it is crucial deepen our understanding into the mechanisms that pathogens use to thrive in complex environments. Most bacteria use sophisticated chemical communication systems to sense their population density and coordinate gene expression in a collective manner, a process that is termed “quorum sensing” (QS). The human pathogen Pseudomonas aeruginosa uses several small molecules to regulate QS, and one of them is N-butyryl-l-homoserine lactone (C₄-HSL). Using an activity-based protein profiling (ABPP) strategy, we designed biomimetic probes with a photoreactive group and a ‘click’ tag as an analytical handle. Using these probes, we have identified previously uncharacterized proteins that are part of the P. aeruginosa QS network, and we uncovered an additional role for this natural autoinducer in the virulence regulon of P. aeruginosa, through its interaction with PhzB1/2 that results in inhibition of pyocyanin production.

Short-chain reactive probes can be used as tools to shed new light on virulence mechanisms in bacterial pathogens.     

Homogentisic acid γ-lactone suppresses the virulence factors of Pseudomonas aeruginosa by quenching its quorum sensing signal molecules

Homogentisic acid γ-lactone exhibited excellent anti-quorum sensing (QS) and anti-biofilm activities against Pseudomonas aeruginosa. Moreover, it suppressed the QS-dependent virulence factors in P. aeruginosa by quenching its QS signal molecules.     

The role of quorum sensing in the pathogenicity of the cunning aggressor Pseudomonas aeruginosa

Recent decades have revealed that many bacterial species are capable of communicating with each other, and this observation has been largely responsible for a paradigm shift in microbiology. Whereas it was previously believed that bacteria lived as individual cells, it is now acknowledged that bacteria preferentially live in communities in the form of primitive organisms in which the behavior of individual cells is coordinated by cell–cell communication, known as quorum sensing (QS). Bacteria use QS for regulation of the processes involved in their interaction with each other, their environment, and, particularly, higher organisms We have focused on Pseudomonas aeruginosa, an opportunistic pathogen producing more than 30 QS-regulated virulence factors. P. aeruginosa causes several types of nosocomial infection, and lung infection in cystic fibrosis (CF) patients. We review the role of QS in the protective mechanisms of P. aeruginosa and show how disruption of the QS can be used as an approach to control this cunning aggressor.     

Controlling the activity of quorum sensing autoinducers with light

Bacteria use a communication system, called quorum sensing (QS), to organize into communities and synchronize gene expression to promote virulence and secure survival. Here we report on a proof-of-principle for externally interfering with this bacterial communication system, using light. By employing photoswitchable small molecules, we were able to photocontrol the QS-related bioluminescence in an Escherichia coli reporter strain, and the expression of target QS genes and pyocyanin production in Pseudomonas aeruginosa.     

Anti-Pathogenic Properties of the Combination of a T3SS Inhibitory Halogenated Pyrrolidone with C-30 Furanone

Antimicrobial resistance is one of the current public health challenges to be solved. The World Health Organization (WHO) has urgently called for the development of strategies to expand the increasingly limited antimicrobial arsenal. The development of anti-virulence therapies is a viable option to counteract bacterial infections with the possibility of reducing the generation of resistance. Here we report on the chemical structures of pyrrolidones DEXT 1–4 (previously identified as furan derivatives) and their anti-virulence activity on Pseudomonas aeruginosa strains. DEXT 1–4 were shown to inhibit biofilm formation, swarming motility, and secretion of ExoU and ExoT effector proteins. Also, the anti-pathogenic property of DEXT-3 alone or in combination with furanone C-30 (quorum sensing inhibitor) or MBX-1641 (type III secretion system inhibitor) was analyzed in a model of necrosis induced by P. aeruginosa PA14. All treatments reduced necrosis; however, only the combination of C-30 50 µM with DEXT-3 100 µM showed significant inhibition of bacterial growth in the inoculation area and systemic dispersion. In conclusion, pyrrolidones DEXT 1–4 are chemical structures capable of reducing the pathogenicity of P. aeruginosa and with the potential for the development of anti-virulence combination therapies.     

Inhibition of Swarming motility of Pseudomonas aeruginosa by Methanol extracts of Alpinia officinarum Hance. and Cinnamomum tamala T. Nees and Eberm

Bacterial drug resistance is a challenge in clinical settings, especially in countries like India. Hence, discovery of novel alternative therapeutics has become a necessity in the fight against drug resistance. Compounds that inhibit bacterial virulence properties form new therapeutic alternatives. Pseudomonas aeruginosa is an opportunistic, nosocomial pathogen that infects immune-compromised patients. Swarming motility is an important virulence property of Pseudomonas which aids it in reaching host cells under nutrient limiting conditions. Here, we report the screening of five plant extracts against swarming motility of P. aeruginosa and show that methanol extracts of Alpinia officinarum and Cinnamomum tamala inhibit swarming motility at 5 μg mL^?1 without inhibiting its growth. These extracts did not inhibit swimming and twitching motilities indicating a mode of action specific to swarming pathway. Preliminary experiments indicated that rhamnolipid production was not affected. This study reveals the potential of the two plants in anti-virulence drug discovery.     

Quorum sensing regulates electric current generation of Pseudomonas aeruginosa PA14 in bioelectrochemical systems

Here, we show that quorum sensing (QS) modulates the current generation of the anode-respiring bacterium Pseudomonas aeruginosa because it controls the production of phenazines, which mediate the electron transfer to the anode. The current generation by a wildtype (WT) strain P. aeruginosa PA14 and the GacS/GacA protein-regulatory mutant retS was investigated under different environmental conditions. The retS mutant generated significantly higher current (45-fold) than the WT under anaerobic conditions. Anaerobic current generation by the WT was 28-fold higher with extraneously supplied lactones (a QS-signaling molecule). Compared to anaerobic conditions, the WT with some oxygen (microaerobic conditions) exhibited enhanced phenazine production (39-fold) and current levels (48-fold). Iron-rich medium and microaerobic conditions had a negative impact on current generation by retS. All these results were directly linked to QS activity in P. aeruginosa, thus, demonstrating the importance of this bacterial communication system for current generation in BESs. We also show that BESs represent a new tool for real-time investigation of phenazine-related QS activity.     

Thiazole Analogues of the Marine Alkaloid Nortopsentin as Inhibitors of Bacterial Biofilm Formation

Anti-virulence strategy is currently considered a promising approach to overcome the global threat of the antibiotic resistance. Among different bacterial virulence factors, the biofilm formation is recognized as one of the most relevant. Considering the high and growing percentage of multi-drug resistant infections that are biofilm-mediated, new therapeutic agents capable of counteracting the formation of biofilms are urgently required. In this scenario, a new series of 18 thiazole derivatives was efficiently synthesized and evaluated for its ability to inhibit biofilm formation against the Gram-positive bacterial reference strains Staphylococcus aureus ATCC 25923 and S. aureus ATCC 6538 and the Gram-negative strain Pseudomonas aeruginosa ATCC 15442. Most of the new compounds showed a marked selectivity against the Gram-positive strains. Remarkably, five compounds exhibited BIC₅₀ values against S. aureus ATCC 25923 ranging from 1.0 to 9.1 µM. The new compounds, affecting the biofilm formation without any interference on microbial growth, can be considered promising lead compounds for the development of a new class of anti-virulence agents.     

Halogenated Dihydropyrrol-2-One Molecules Inhibit Pyocyanin Biosynthesis by Blocking the Pseudomonas Quinolone Signaling System

Quorum-sensing (QS) systems of Pseudomonas aeruginosa are involved in the control of biofilm formation and virulence factor production. The current study evaluated the ability of halogenated dihydropyrrol-2-ones (DHP) (Br (4a), Cl (4b), and F (4c)) and a non-halogenated version (4d) to inhibit the QS receptor proteins LasR and PqsR. The DHP molecules exhibited concentration-dependent inhibition of LasR and PqsR receptor proteins. For LasR, all compounds showed similar inhibition levels. However, compound 4a (Br) showed the highest decrease (two-fold) for PqsR, even at the lowest concentration (12.5 µg/mL). Inhibition of QS decreased pyocyanin production amongst P. aeruginosa PAO1, MH602, ATCC 25619, and two clinical isolates (DFU-53 and 364707). In the presence of DHP, P. aeruginosa ATCC 25619 showed the highest decrease in pyocyanin production, whereas clinical isolate DFU-53 showed the lowest decrease. All three halogenated DHPs also reduced biofilm formation by between 31 and 34%. The non-halogenated compound 4d exhibited complete inhibition of LasR and had some inhibition of PqsR, pyocyanin, and biofilm formation, but comparatively less than halogenated DHPs.     

Inhibition of Quorum Sensing,Motility and Biofilm Formation of Pseudomonas aeruginosa by Copper Oxide Nanostructures

Quorum sensing (QS) is the communication between bacterial cells governed by their population density and regulated by the genes controlling virulence factors and biofilm formation. Multiple mechanisms of biofilms are resistive to antimicrobial chemotherapy; therefore novel strategies are required to overcome its limitations. Here, we report the effect of various copper oxide nanostructures (CuO-NSs) on quorum sensing inhibition. The two-dimensional CuO-NSs such as interlaced nanodiscs, nanodiscs and leaf-shaped nanosheets are prepared via a simple chemical method. The Quorum sensing inhibition (QSI) activity of all the CuO-NS are examined using reporter strain Chromobacterium violaceum CV026 and Escherichia coli pSB1142. We found that the CuO-interlaced nanodisc structures exhibit better QSI activity than nanodiscs and leaf-shaped sheets. The interlaced nanodisc structures are inhibited various long-chain N-acyl homoserine lactones (AHLs) mediated QS individually and confirmed by other QS-associated phenomena for Pseudomonas aeruginosa, including biofilm inhibition, inhibition of virulence factors such as pyocyanin, protease production and swarming motility. Thus QSI activity of CuO-NSs is solely dependent on specific shape offering large surface area and more active sites. The CuO-NS is effective quorum sensing inhibitors, which has potential clinical applications in the management of P. aeruginosa associated infections.

     

Computational discovery of putative quorum sensing inhibitors against LasR and RhlR receptor proteins of Pseudomonas aeruginosa

Drugs have been discovered in the past mainly either by identification of active components from traditional remedies or by unpredicted discovery. A key motivation for the study of structure based virtual screening is the exploitation of such information to design targeted drugs. In this study, structure based virtual screening was used in search for putative quorum sensing inhibitors (QSI) of Pseudomonas aeruginosa. The virtual screening programme Glide version 5.5 was applied to screen 1,920 natural compounds/drugs against LasR and RhlR receptor proteins of P. aeruginosa. Based on the results of in silico docking analysis, five top ranking compounds namely rosmarinic acid, naringin, chlorogenic acid, morin and mangiferin were subjected to in vitro bioassays against laboratory strain PAO1 and two more antibiotic resistant clinical isolates, P. aeruginosa AS1 (GU447237) and P. aeruginosa AS2 (GU447238). Among the five compounds studied, except mangiferin other four compounds showed significant inhibition in the production of protease, elastase and hemolysin. Further, all the five compounds potentially inhibited the biofilm related behaviours. This interaction study provided promising ligands to inhibit the quorum sensing (QS) mediated virulence factors production in P. aeruginosa.     

Lemon Oils Attenuate the Pathogenicity of Pseudomonas aeruginosa by Quorum Sensing Inhibition

The chemical composition of three Citrus limon oils: lemon essential oil (LEO), lemon terpenes (LT) and lemon essence (LE), and their influence in the virulence factors production and motility (swarming and swimming) of two Pseudomonas aeruginosa strains (ATCC 27853 and a multidrug-resistant HT5) were investigated. The main compound, limonene, was also tested in biological assays. Eighty-four compounds, accounting for a relative peak area of 99.23%, 98.58% and 99.64%, were identified by GC/MS. Limonene (59–60%), γ-terpinene (10–11%) and β-pinene (7–15%) were the main compounds. All lemon oils inhibited specific biofilm production and bacterial metabolic activities into biofilm in a dose-dependent manner (20–65%, in the range of 0.1–4 mg mL⁻¹) of both strains. Besides, all samples inhibited about 50% of the elastase activity at 0.1 mg mL⁻¹. Pyocyanin biosynthesis decreases until 64% (0.1–4 mg mL⁻¹) for both strains. Swarming motility of P. aeruginosa ATCC 27853 was completely inhibited by 2 mg mL⁻¹ of lemon oils. Furthermore, a decrease (29–55%, 0.1–4 mg mL⁻¹) in the synthesis of Quorum sensing (QS) signals was observed. The oils showed higher biological activities than limonene. Hence, their ability to control the biofilm of P. aeruginosa and reduce the production of virulence factors regulated by QS makes lemon oils good candidates to be applied as preservatives in the food processing industry.     

Juglone Inactivates Pseudomonas aeruginosa through Cell Membrane Damage,Biofilm Blockage,and Inhibition of Gene Expression

Due to the strong drug resistance of Pseudomonas aeruginosa (P. aeruginosa), the inhibition effects of conventional disinfectants and antibiotics are not obvious. Juglone extracted from discarded walnut husk, as a kind of plant-derived antimicrobial agent, has the advantages of naturalness, high efficiency, and low residue, with a potential role in the inhibition of P. aeruginosa. This study elucidated the inhibitory effect of juglone on the growth of plankton and the formation of P. aeruginosa biofilm. The results showed that juglone (35 μg/mL) had an irreversible inhibitory effect on P. aeruginosa colony formation (about 10⁷ CFU/mL). The integrity and permeability of the cell membrane were effectively destroyed, accompanied by disorder of the membrane permeability, mass leakage of the cytoplasm, and ATP consumption. Further studies manifested that juglone could induce the abnormal accumulation of ROS in cells and block the formation of the cell membrane. In addition, RT-qPCR showed that juglone could effectively block the expression of five virulence genes and two genes involved in the production of extracellular polymers, thereby reducing the toxicity and infection of P. aeruginosa and preventing the production of extracellular polymers. This study can provide support for the innovation of antibacterial technology toward P. aeruginosa in food.     

Highly Stable,Amide‐Bridged Autoinducing Peptide Analogues that Strongly Inhibit the AgrC Quorum Sensing Receptor in Staphylococcus aureus

Blocking quorum sensing (QS) pathways has attracted considerable interest as an approach to suppress virulence in bacterial pathogens. Toward this goal, we recently developed analogues of a native autoinducing peptide (AIP‐III) signal that can inhibit AgrC‐type QS receptors and attenuate virulence phenotypes in Staphylococcus aureus. Application of these compounds is limited, however, as they contain hydrolytically unstable thioester linkages and have only low aqueous solubilities. Herein, we report amide‐linked AIP analogues with greatly enhanced hydrolytic stabilities and solubilities relative to our prior analogues, whilst maintaining strong potencies as AgrC receptor inhibitors in S. aureus. These compounds represent powerful tools for the study of QS.     

Highly Stable,Amide‐Bridged Autoinducing Peptide Analogues that Strongly Inhibit the AgrC Quorum Sensing Receptor in Staphylococcus aureus

Blocking quorum sensing (QS) pathways has attracted considerable interest as an approach to suppress virulence in bacterial pathogens. Toward this goal, we recently developed analogues of a native autoinducing peptide (AIP‐III) signal that can inhibit AgrC‐type QS receptors and attenuate virulence phenotypes in Staphylococcus aureus. Application of these compounds is limited, however, as they contain hydrolytically unstable thioester linkages and have only low aqueous solubilities. Herein, we report amide‐linked AIP analogues with greatly enhanced hydrolytic stabilities and solubilities relative to our prior analogues, whilst maintaining strong potencies as AgrC receptor inhibitors in S. aureus. These compounds represent powerful tools for the study of QS.