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.     

Inhibition of Quorum-Sensing Regulator from Pseudomonas aeruginosa Using a Flavone Derivative

Quorum sensing (QS) is a cell-to-cell communication process that controls bacterial collective behaviors. The QS network regulates and coordinates bacterial virulence factor expression, antibiotic resistance and biofilm formation. Therefore, inhibition of the QS system is an effective strategy to suppress the bacterial virulence. Herein, we identify a phosphate ester derivative of chrysin as a potent QS inhibitor of the human pathogen Pseudomonas aeruginosa (P. aeruginosa) using a designed luciferase reporter assay. In vitro biochemical analysis shows that the chrysin derivative binds to the bacterial QS regulator LasR and abrogates its DNA-binding capability. In particular, the derivative exhibits higher anti-virulence activity compared to the parent molecule. All the results reveal the potential application of flavone derivative as an anti-virulence compound to combat the infectious diseases caused by P. aeruginosa.     

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.     

Fluorogenic probes for detecting deacylase and demethylase activity towards post-translationally-modified lysine residues

Reversible enzymatic post-translational modification of the ε-amino groups of lysine residues (e.g. N-acylation reactions) plays an important role in regulating the cellular activities of numerous proteins. This study describes how enzyme catalyzed N-deprotection of lysine residues of non-fluorescent peptide-coumarin probes can be used to generate N-deprotected peptides that undergo spontaneous O- to N-ester transfer reactions (uncatalyzed) to generate a highly fluorescent N-carbamoyl peptide. This enables detection of enzyme catalyzed N-deacetylation, N-demalonylation, N-desuccinylation and N-demethylation reactions activities towards the N-modified lysine residues of these probes using simple ‘turn on’ fluorescent assays.

We developed “turn-on” fluorescent probes that detect enzymatic lysine deacylation and demethylation critical for epigenetic and other cellular phenomena, using intramolecular O- to N-ester transfer reactions.     

Chemoproteomic profiling of itaconations in Salmonella

Itaconate is an immunoregulatory and anti-bacterial metabolite, and plays important roles in host–pathogen interactions. Chemoproteomic strategies have been used to explore the anti-inflammatory effects of itaconate on activated macrophages and it has been found that many key proteins in immune pathways were modified; however, how itaconate modulates pathogens was not fully understood. Here, we have designed and synthesized a series of itaconate-based bioorthogonal probes, which enable quantitative and site-specific profiling of itaconated proteins and sites in Salmonella. Among many proteins related to energy metabolism, we identified a key enzyme involved in the glyoxylate cycle, isocitrate lyase (ICL), as the most prominent target. Covalent modification of the active-site cysteine in ICL by itaconate abolishes the enzyme activity and suppresses bacterial growth. Our chemoproteomic study has uncovered the wide array of itaconation targets in Salmonella and provided a comprehensive resource for understanding the anti-bacterial function of this intriguing metabolite.

Bioorthogonal probes have been developed to enable quantitative and site-specific profiling of itaconate modifications in Salmonella.     

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.     

Pinkment: a synthetic platform for the development of fluorescent probes for diagnostic and theranostic applications

Reaction-based fluorescent-probes have proven successful for the visualisation of biological species in various cellular processes. Unfortunately, in order to tailor the design of a fluorescent probe to a specific application (i.e. organelle targeting, material and theranostic applications) often requires extensive synthetic efforts and the synthetic screening of a range of fluorophores to match the required synthetic needs. In this work, we have identified Pinkment-OH as a unique “plug-and-play” synthetic platform that can be used to develop a range of ONOO⁻ responsive fluorescent probes for a variety of applications. These include theranostic-based applications and potential material-based/bioconjugation applications. The as prepared probes displayed an excellent sensitivity and selectivity for ONOO⁻ over other ROS. In vitro studies using HeLa cells and RAW 264.7 macrophages demonstrated their ability to detect exogenously and endogenously produced ONOO⁻. Evaluation in an LPS-induced inflammation mouse model illustrated the ability to monitor ONOO⁻ production in acute inflammation. Lastly, theranostic-based probes enabled the simultaneous evaluation of indomethacin-based therapeutic effects combined with the visualisation of an inflammation biomarker in RAW 264.7 cells.

Pinkment, a resorufin based ONOO⁻ selective and sensitive ‘plug and play’ fluorescence-based platform for in vitro and in vivo use, enables facile functionalisation for various imaging and theranostic applications.     

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.     

Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high in vivo efficacy

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid''s antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL⁻¹, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 μg mL⁻¹). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid''s N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.

Structure–activity relationship studies of the natural product cystobactamid at four different positions led to novel imaging probes and analogs with superior antibacterial activities and in vivo efficacy.     

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.     

2-Amino-3′-dialkylaminobiphenyl-based fluorescent intracellular probes for nitric oxide surrogate N2O3

Fluorescent probes for nitric oxide (NO), or more frequently for its oxidized surrogate dinitrogen trioxide (N₂O₃), have enabled scientists to study the contributions of this signaling molecule to many physiological processes. Seeking to improve upon limitations of other probes, we have developed a family of fluorescent probes based on a 2-amino-3′-dialkylaminobiphenyl core. This core condenses with N₂O₃ to form benzo[c]cinnoline structures, incorporating the analyte into the newly formed fluorophore, which results in product fluorescence with virtually no background contribution from the initial probe. We varied the substituents in the core in order to optimize both the reactivity of the probes with N₂O₃ and their cinnoline products'' fluorescence wavelengths and brightness. The top candidates were then applied to cultured cells to verify that they could respond to NO within cellular milieus, and the top performer, NO₅₃₀, was compared with a “gold standard” commercial probe, DAF-FM, in a macrophage-derived cell line, RAW 264.7, stimulated to produce NO. NO₅₃₀ demonstrated similar or better sensitivity and higher selectivity for NO than DAF, making it an attractive potential alternative for NO tracking in various applications.

A thorough structure-property relationship study has produced much-improved fluorescence probes for nitric oxide surrogate, e.g. N₂O₃, compared to the original NO₅₅₀.     

Long-lived lanthanide emission via a pH-sensitive and switchable LRET complex

Lanthanide-based luminescence resonance energy transfer (LRET) can be used as a tool to enhance lanthanide emission for time-resolved cellular imaging applications. By shortening lanthanide emission lifetimes whilst providing an alternative radiative pathway to the formally forbidden, weak lanthanide-only emission, the photon flux of such systems is increased. With this aim in mind, we investigated energy transfer in differently spaced donor–acceptor terbium–rhodamine pairs with the LRET “on” (low pH) and LRET “off” (high pH). Results informed the design, preparation and characterisation of a compound containing terbium, a spectrally-matched pH-responsive fluorophore and a receptor-targeting group. By combining these elements, we observed switchable LRET, where the targeting group sensitises lanthanide emission, resulting in an energy transfer to the rhodamine dye with an efficiency of E = 0.53. This strategy can be used to increase lanthanide emission rates for brighter optical probes.

A pH-sensitive luminescence resonance energy transfer (LRET) was explored as a method to increase photon flux in a terbium-rhodamine-receptor targeting group construct. At low pH, long-lived dye emission and shorter terbium lifetimes were observed.     

Attention-based generative models for de novo molecular design

Attention mechanisms have led to many breakthroughs in sequential data modeling but have yet to be incorporated into any generative algorithms for molecular design. Here we explore the impact of adding self-attention layers to generative β-VAE models and show that those with attention are able to learn a complex “molecular grammar” while improving performance on downstream tasks such as accurately sampling from the latent space (“model memory”) or exploring novel chemistries not present in the training data. There is a notable relationship between a model''s architecture, the structure of its latent memory and its performance during inference. We demonstrate that there is an unavoidable tradeoff between model exploration and validity that is a function of the complexity of the latent memory. However, novel sampling schemes may be used that optimize this tradeoff. We anticipate that attention will play an important role in future molecular design algorithms that can make efficient use of the detailed molecular substructures learned by the transformer.

An implementation of attention within the variational autoencoder framework for continuous representation of molecules. The addition of attention significantly increases model performance for complex tasks such as exploration of novel chemistries.     

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.     

Assessing long-range contributions to the charge asymmetry of ion adsorption at the air–water interface

Anions generally associate more favorably with the air–water interface than cations. In addition to solute size and polarizability, the intrinsic structure of the unperturbed interface has been discussed as an important contributor to this bias. Here we assess quantitatively the role that intrinsic charge asymmetry of water''s surface plays in ion adsorption, using computer simulations to compare model solutes of various size and charge. In doing so, we also evaluate the degree to which linear response theory for solvent polarization is a reasonable approach for comparing the thermodynamics of bulk and interfacial ion solvation. Consistent with previous works on bulk ion solvation, we find that the average electrostatic potential at the center of a neutral, sub-nanometer solute at the air–water interface depends sensitively on its radius, and that this potential changes quite nonlinearly as the solute''s charge is introduced. The nonlinear response closely resembles that of the bulk. As a result, the net nonlinearity of ion adsorption is weaker than in bulk, but still substantial, comparable to the apparent magnitude of macroscopically nonlocal contributions from the undisturbed interface. For the simple-point-charge model of water we study, these results argue distinctly against rationalizing ion adsorption in terms of surface potentials inherent to molecular structure of the liquid''s boundary.

Cations and anions have different affinities for the air-water interface. The intrinsic orientation of surface molecules suggests such an asymmetry, but the bias is dominated by solvent response that is spatially local and significantly nonlinear.

Counter to expectations from conventional theories of solvation, there is a large body of both computational and experimental evidence indicating that small ions can adsorb to the air–water interface.^1–9 Implications across the biological, atmospheric and physical sciences have inspired efforts to understand the microscopic driving forces for ions associating with hydrophobic interfaces in general.^10–21 A particular emphasis has been placed on understanding ion specificity, i.e., why some ions exhibit strong interfacial affinity while others do not. Empirical trends indicate that ion size and polarizability are important factors, as could be anticipated from conventional theory. More surprisingly, the sign of a solute''s charge can effect a significant bias, with anions tending to adsorb more favorably than cations.Here we examine the microscopic origin of this charge asymmetry in interfacial ion adsorption. We specifically assess whether the thermodynamic preference can be simply and generally understood in terms of long-range biases that are intrinsic to an aqueous system surrounded by vapor. By “long-range” and “nonlocal” we refer to macroscopically large scales, i.e., collective forces that are felt at arbitrarily long distance. Such a macroscopically long-range bias is expected from the air–water interface due to its average polarization, and by some measures the bias is quite strong. By contrast, “local” contributions comprise the entire influence of a solute''s microscopic environment, including electrostatic forces from molecules that are many solvation shells away – any influence that decays over a sub-macroscopic length scale.The importance of macroscopically nonlocal contributions has been discussed extensively in the context of ion solvation in bulk liquid water, which we review in Section 1 as a backdrop for interfacial solvation. The notion that such contributions strongly influence charge asymmetry of solvation at the air–water interface has informed theoretical approaches and inspired criticism of widely used force fields for molecular simulation.^22,23 A full understanding of their role in interfacial adsorption, however, is lacking.In the course of this study, we will also evaluate the suitability of dielectric continuum theory (DCT) to describe the adsorption process. DCT has provided an essential conceptual framework for rationalizing water''s response to electrostatic perturbations. But a more precise understanding of its applicability is needed, particularly for the construction of more elaborate models (e.g., with heterogeneous polarizability near interfaces^24–26) and for the application of DCT to evermore complex (e.g., nanoconfined^27,28) environments.