Small-molecule modulators of Listeria monocytogenes biofilm development

Listeria monocytogenes is an important food-borne pathogen whose ability to form disinfectant-tolerant biofilms on a variety of surfaces presents a food safety challenge for manufacturers of ready-to-eat products. We developed here a high-throughput biofilm assay for L. monocytogenes and, as a proof of principle, used it to screen an 80-compound protein kinase inhibitor library to identify molecules that perturb biofilm development. The screen yielded molecules toxic to multiple strains of Listeria at micromolar concentrations, as well as molecules that decreased (≤ 50% of vehicle control) or increased (≥ 200%) biofilm formation in a dose-dependent manner without affecting planktonic cell density. Toxic molecules-including the protein kinase C antagonist sphingosine-had antibiofilm activity at sub-MIC concentrations. Structure-activity studies of the biofilm inhibitory compound palmitoyl-d,l-carnitine showed that while Listeria biofilm formation was inhibited with a 50% inhibitory concentration of 5.85 ± 0.24 μM, d,l-carnitine had no effect, whereas palmitic acid had stimulatory effects. Saturated fatty acids between C(9:0) and C(14:0) were Listeria biofilm inhibitors, whereas fatty acids of C(16:0) or longer were stimulators, showing chain length specificity. De novo-synthesized short-chain acyl carnitines were less effective biofilm inhibitors than the palmitoyl forms. These molecules, whose activities against bacteria have not been previously established, are both useful probes of L. monocytogenes biology and promising leads for the further development of antibiofilm strategies.     

Inhibition of the LOX enzyme family members with old and new ligands. Selectivity analysis revisited

Lysyl oxidase (LOX) enzymes as potential drug targets maintain constant attention in the therapy of fibrosis, cancer and metastasis. In order to measure the inhibitory activity of small molecules on the LOX enzyme family members a fluorometric activity screening method was developed. During assay validation, previously reported non-selective small inhibitor molecules (BAPN, MCP-1, thiram, disulfiram) were investigated on all of the major LOX enzymes. We confirmed that MCP-1, thiram, disulfiram are in fact pan-inhibitors, while BAPN inhibits only LOX-like enzymes (preferably LOX-like-protein-2, LOXL2) in contrast to the previous reports. We measured the LOX inhibitory profile of a small targeted library generated by 2D ligand-based chemoinformatics methods. Ten hits (10.4% hit rate) were identified, and the compounds showed distinct activity profiles. Potential inhibitors were also identified for LOX-like-protein-3 (LOXL3) and LOX-like-protein-4 (LOXL4), that are considered as emerging drug targets in the therapy of melanoma and gastric cancer.     

Novel Multiscale Modeling Tool Applied to Pseudomonas aeruginosa Biofilm Formation

Multiscale modeling is used to represent biological systems with increasing frequency and success. Multiscale models are often hybrids of different modeling frameworks and programming languages. We present the MATLAB-NetLogo extension (MatNet) as a novel tool for multiscale modeling. We demonstrate the utility of the tool with a multiscale model of Pseudomonas aeruginosa biofilm formation that incorporates both an agent-based model (ABM) and constraint-based metabolic modeling. The hybrid model correctly recapitulates oxygen-limited biofilm metabolic activity and predicts increased growth rate via anaerobic respiration with the addition of nitrate to the growth media. In addition, a genome-wide survey of metabolic mutants and biofilm formation exemplifies the powerful analyses that are enabled by this computational modeling tool.     

群体感应抑制剂对海洋生态功能菌生物膜形成的影响

[目的]研究天然群体感应抑制剂(Quorum sensing inhibitors,QSI)分子对海洋生态功能菌生物膜形成的影响.[方法]以对污损生物幼虫附着具有诱导作用的海洋细菌为目标菌,通过在其生物膜的形成过程中添加天然群体感应抑制剂,研究其对目标菌成膜细菌数和浮游细菌数、生物膜形态以及生物膜表面胞外多糖含量的影响.[结果]呋喃酮和吡啶在50 mg/L时,对8株目标菌的成膜有显著的抑制作用,抑制率在80％左右,吲哚、青霉烷酸和香豆素在较高浓度800 mg/L才有比较好的抑制活性.生长抑制实验结果显示,同等浓度下,QSI分子对目标菌成膜的抑制活性明显高于其对浮游细菌生长的抑制活性.结果表明,QSI分子主要通过干扰目标菌群体感应系统以抑制生物膜的形成.[结论]研究证实QSI分子在海洋菌生物膜形成过程中具有一定的调控作用.通过添加QSI可能能够间接抑制由生物膜诱导的污损生物附着,从而以新的角度研制新型抗污损物质.     

Experimental and statistical methods to evaluate antibacterial activity of a quaternary pyridinium salt on planktonic,biofilm-forming,and biofilm states

Robust evaluation and comparison of antimicrobial technologies are critical to improving biofilm prevention and treatment. Herein, a multi-pronged experimental framework and statistical models were applied to determine the effects of quaternary pyridinium salt, 4-acetyl-1-hexadecylpyridin-1-ium iodide (QPS-1), on Streptococcus mutans in the planktonic, biofilm-forming and biofilm cell states. Minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) were determined via common methods with novel application of statistical approaches combining random effects models and interval censored data to estimate uncertainties. The MICs and MBCs for planktonic and biofilm-forming states ranged from 3.12 to 12.5 μg ml^?1, with biofilm values only ≈ 8 times higher. Potent anti-biofilm activity and reactive structural features make QPS-1 a promising antibacterial additive for dental and potentially other biomedical devices. Together, the experimental framework and statistical models provide estimates and uncertainties for effective antimicrobial concentrations in multiple cell states, enabling statistical comparisons and improved characterization of antibacterial agents.     

High throughput screening for small molecule enhancers of the interferon signaling pathway to drive next-generation antiviral drug discovery

Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z'>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-μM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit.     

Novel 2, 8-bit derivatives of quinolines attenuate Pseudomonas aeruginosa virulence and biofilm formation

Signal molecules are stimulators of multiple quroum-sensing virulence and biofilm formation. Small molecule analogues have been suspected as a potent inhibitor in therapeutic strategy. Herein, we synthesized a series of small molecule compounds from the 2, 8-bit derivatives of quinoline by Suzuki coupling reaction. We found that these compounds have the biofilm inhibitory effect in normal condition instead of phosphate limitation state. Furthermore, lacZ reporter strain assay and rhamnolipids as well as pyocyanin experiments showed that these compounds did not affect las and pqs system but reduced the expression of rhl. All these results suggest that quinoline derivatives can be treated as potent inhibitors against biofilm and reduce virulence through the rhl system. This research will be useful in designing new quorum sensing inhibitors to attenuate the infection of bacteria.     

In vitro biofilm models to study dental caries: a systematic review

The aim of this systematic review is to characterize and discuss key methodological aspects of in vitro biofilm models for caries-related research and to verify the reproducibility and dose-response of models considering the response to anti-caries and/or antimicrobial substances. Inclusion criteria were divided into Part I (PI): an in vitro biofilm model that produces a cariogenic biofilm and/or caries-like lesions and allows pH fluctuations; and Part II (PII): models showing an effect of anti-caries and/or antimicrobial substances. Within PI, 72.9% consisted of dynamic biofilm models, while 27.1% consisted of batch models. Within PII, 75.5% corresponded to dynamic models, whereas 24.5% corresponded to batch models. Respectively, 20.4 and 14.3% of the studies reported dose-response validations and reproducibility, and 32.7% were classified as having a high risk of bias. Several in vitro biofilm models are available for caries-related research; however, most models lack validation by dose-response and reproducibility experiments for each proposed protocol.     

Multiple receptor conformation docking and dock pose clustering as tool for CoMFA and CoMSIA analysis – a case study on HIV-1 protease inhibitors

Multiple receptors conformation docking (MRCD) and clustering of dock poses allows seamless incorporation of receptor binding conformation of the molecules on wide range of ligands with varied structural scaffold. The accuracy of the approach was tested on a set of 120 cyclic urea molecules having HIV-1 protease inhibitory activity using 12 high resolution X-ray crystal structures and one NMR resolved conformation of HIV-1 protease extracted from protein data bank. A cross validation was performed on 25 non-cyclic urea HIV-1 protease inhibitor having varied structures. The comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models were generated using 60 molecules in the training set by applying leave one out cross validation method, r_loo² values of 0.598 and 0.674 for CoMFA and CoMSIA respectively and non-cross validated regression coefficient r² values of 0.983 and 0.985 were obtained for CoMFA and CoMSIA respectively. The predictive ability of these models was determined using a test set of 60 cyclic urea molecules that gave predictive correlation (r_pred²) of 0.684 and 0.64 respectively for CoMFA and CoMSIA indicating good internal predictive ability. Based on this information 25 non-cyclic urea molecules were taken as a test set to check the external predictive ability of these models. This gave remarkable out come with r_pred² of 0.61 and 0.53 for CoMFA and CoMSIA respectively. The results invariably show that this method is useful for performing 3D QSAR analysis on molecules having different structural motifs.     

Molecular mechanisms of compounds affecting bacterial biofilm formation and dispersal

Bacteria can switch between planktonic forms (single cells) and biofilms, i.e., bacterial communities growing on solid surfaces and embedded in a matrix of extracellular polymeric substance. Biofilm formation by pathogenic bacteria often results in lower susceptibility to antibiotic treatments and in the development of chronic infections; thus, biofilm formation can be considered an important virulence factor. In recent years, much attention has been directed towards understanding the biology of biofilms and towards searching for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we review selected examples of target-based screening for anti-biofilm agents: We focus on inhibitors of quorum sensing, possibly the most characterized target for molecules with anti-biofilm activity, and on compounds interfering with the metabolism of the signal molecule cyclic di-GMP metabolism and on inhibitors of DNA and nucleotide biosynthesis, which represent a novel and promising class of biofilm inhibitors. Finally, we discuss the activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds.     

Chemoinformatics for rational discovery of safe antibacterial drugs: Simultaneous predictions of biological activity against streptococci and toxicological profiles in laboratory animals

Streptococci are a group of Gram-positive bacteria which are responsible for causing many diverse diseases in humans and other animals worldwide. The high prevalence of resistance of these bacteria to current antibacterial drugs is an alarming problem for the scientific community. The battle against streptococci by using antimicrobial chemotherapies will depend on the design of new chemicals with high inhibitory activity, having also as low toxicity as possible. Multi-target approaches based on quantitative–structure activity relationships (mt-QSAR) have played a very important role, providing a better knowledge about the molecular patterns related with the appearance of different pharmacological profiles including antimicrobial activity. Until now, almost all mt-QSAR models have considered the study of biological activity or toxicity separately. In the present study, we develop by the first time, a unified multitasking (mtk) QSAR model for the simultaneous prediction of anti-streptococci activity and toxic effects against biological models like Mus musculus and Rattus norvegicus. The mtk-QSAR model was created by using artificial neural networks (ANN) analysis for the classification of compounds as positive (high biological activity and/or low toxicity) or negative (otherwise) under diverse sets of experimental conditions. Our mtk-QSAR model, correctly classified more than 97% of the cases in the whole database (more than 11,500 cases), serving as a promising tool for the virtual screening of potent and safe anti-streptococci drugs.     

Pharmacophore modeling and virtual screening for designing potential 5-Lipoxygenase inhibitors

Inhibitors of the 5-Lipoxygenase (5-LOX) pathway have a therapeutic potential in a variety of inflammatory disorders such as asthma. In this study, chemical feature based pharmacophore models of inhibitors of 5-LOX have been developed with the aid of HipHop and HypoGen modules within Catalyst program package. The best quantitative pharmacophore model, Hypo1, which has the highest correlation coefficient (0.97), consists of two hydrogen-bond acceptors, one hydrophobic feature and one ring aromatic feature. Hypo1 was further validated by test set and cross validation method. The application of the model shows great success in predicting the activities of 65 known 5-LOX inhibitors in our test set with a correlation coefficient of 0.85 with a cross validation of 95% confidence level, proving that the model is reliable in identifying structurally diverse compounds for inhibitory activity against 5-LOX. Furthermore, Hypo1 was used as a 3D query for screening Maybridge and NCI databases within catalyst and also drug like compounds obtained from Enamine Ltd, which follow Lipinski’s rule of five. The hit compounds were subsequently subjected to filtering by docking and visualization, to identify the potential lead molecules. Finally 5 potential lead compounds, identified in the above process, were evaluated for their inhibitory activities. These studies resulted in the identification of two compounds with potent inhibition of 5-LOX activity with IC₅₀ of 14 μM and 35 μM, respectively. These studies thus validate the pharmacophore model generated and suggest the usefulness of the model in screening of various small molecule libraries and identification of potential lead compounds for 5-LOX inhibition.     

A thin film electro-acoustic enzyme biosensor allowing the detection of trace organophosphorus pesticides

We report an analytical method using a thin film electro-acoustic resonator for the detection of organophosphorus pesticides. The acetylcholinesterase (AChE) enzyme was immobilized on the surface of the resonator. In the presence of organophosphorus compounds, the degree of inhibitory effect of organophosphorus compounds on the AChE activity and the concentration of pesticides were detected in real time by measuring the frequency shift of the resonator. The proposed device has a remarkably low detection limit of 1.8×10(-11)M and obvious advantages such as small size, simple operation, and integrated circuit compatibility, providing a promising tool for pesticide analysis.     

Reduction of antibiotic-induced biofilm accumulation of Pseudomonas aeruginosa by quaternized phytoglycogen

Biofilms are oft cited as a factor in the unwanted persistence and recalcitrance of microbial life and a strong research initiative exists to identify, understand, and target vulnerabilities. Phytoglycogen is a biodegradable nanoparticulate biomaterial that is purified from crop plants. Importantly, the highly branched glucan structure provides a scaffold on which to construct novel polymers. Functionalized phytoglycogen (FP) was synthesized using green chemistry principles. Screening of several molecules identified a form of quaternized phytoglycogen which reduced biofilm formation and accretion by Pseudomonas aeruginosa. Exposing P. aeruginosa to modified phytoglycogen and antibiotic in combination not only substantively reduced biofilms, but also prevented increased biofilm formation, a biological response to suboptimal antibiotic concentrations. Treatment of pregrown biofilms with sub-minimum inhibitory concentration antibiotic alone also led to increased proliferation, whereas FP-antibiotic combinations prevented or reduced the extent of this. Swimming, swarming and twitching motility, all critical for biofilm development, were negatively affected by FP. This work supports phytoglycogen as a promising foundational molecule for novel polymers, including those with anti-biofilm function. Critically, in addition to published reports on how suboptimal antibiotic concentrations promote biofilm formation, we demonstrated a similar effect upon pre-existing biofilms, indicating a further route for the failure of antibiotic therapies.     

A microfluidic gradient mixer-flow chamber as a new tool to study biofilm development under defined solute gradients

Understanding the dynamics of biofilm development in response to chemical cues and signals is required toward the development of controllable biofilm-mediated bioprocesses. In this study, we report a new biofilm growth system that integrates a microfluidic gradient mixer with a biofilm growth chamber. The biofilm growth system allows biofilms to grow under defined solute gradients and enables nondestructive monitoring of the biofilm development dynamics in response to the defined gradients. The solute gradients generated in the system were simulated and then validated experimentally. We then demonstrated the applicability of the biofilm growth system in studying biofilm development under defined solute gradients. Specifically, we examined biofilm development of Shewanella oneidensis and Comamonas testosteroni under a defined calcium and nitrate gradient, respectively. Using two C. testosteroni strains (WDL7 and I2), we further demonstrated the applicability of our biofilm growth system to study the development of coculture biofilms under a defined solute gradient. Our results show that the biofilm growth system we have developed here can be a promising tool to reveal the dynamics of biofilm development in response to chemical cues and signals as well as the interorganism interactions in coculture biofilms.