Flavones as Quorum Sensing Inhibitors Identified by a Newly Optimized Screening Platform Using Chromobacterium violaceum as Reporter Bacteria.

Quorum sensing (QS) is the process by which bacteria produce and detect signal molecules to coordinate their collective behavior. This intercellular communication is a relevant target for anti-biofilm therapies. Here we have optimized a screening-applicable assay to search for new quorum sensing inhibitors from natural compound libraries. In this system, QS is correlated with the production of violacein, which is directly controlled by the LuxI/LuxR system in Chromobacterium violaceum ATCC 31532. The parallel use of C. violaceum Tn5-mutant CV026, which depends on auto-inducer addition, allows simultaneous discrimination of compounds that act as quenchers of the AHL signal (quorum quenchers). The incorporation of a redox stain into the platform allowed further distinction between QS inhibitors, quorum quenchers and antibacterial compounds. A pilot screening was performed with 465 natural and synthetic flavonoids. All the most active compounds were flavones and they displayed potencies (IC50) in the range of 3.69 to 23.35 µM. These leads were particularly promising as they inhibited the transition from microcolonies into mature biofilms from Escherichia coli and Pseudomonas aeruginosa strains. This approach can be very effective in identifying new antimicrobials posing lesser risks of resistance.

Amphipathic ß2,2-Amino Acid Derivatives Suppress Infectivity and Disrupt the Intracellular Replication Cycle of Chlamydia pneumoniae.

We demonstrate in the current work that small cationic antimicrobial ß2,2-amino acid derivatives (Mw < 500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (< 5 µM, i.e. < 3.4 µg/mL). C. pneumoniae is an atypical respiratory pathogen associated with frequent treatment failures and persistent infections. This gram-negative bacterium has a biphasic life cycle as infectious elementary bodies and proliferating reticulate bodies, and efficient treatment is challenging because of its long and obligate intracellular replication cycle within specialized inclusion vacuoles. Chlamydicidal effect of the ß2,2-amino acid derivatives in infected human epithelial cells was confirmed by transmission electron microscopy. Images of infected host cells treated with our lead derivative A2 revealed affected chlamydial inclusion vacuoles 24 hours post infection. Only remnants of elementary and reticulate bodies were detected at later time points. Neither the EM studies nor resazurin-based cell viability assays showed toxic effects on uninfected host cells or cell organelles after A2 treatment. Besides the effects on early intracellular inclusion vacuoles, the ability of these ß2,2-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic ß2,2-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-to-treat intracellular pathogen.

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Mint Flavorings from Candies Inhibit the Infectivity of Chlamydia pneumoniae.

The impact of solvent extracts from the distillation water (flavoring extracts) isolated from mint flavored candies on the infectivity of the intracellular bacterium Chlamydia pneumoniae was evaluated by an in vitro model of epithelial cell infections., The mint flavoring extracts were isolated from the candies by simultaneous hydrodistillation and their chemical composition, established by GC-MS, demonstrated menthol and limonene as the most abundant components. Results obtained by treating C. pneumoniae elementary bodies (EBs) with the flavoring extracts or pure reference compounds showed a significant decrease in EB infectivity, achieved with most of the extracts. This antichlamydial activity could be related to the relatively high menthol content of the extracts. Overall, the obtained data indicates that the flavorings present in the candies are able to target the metabolically quiet, non-replicating form of the bacterium and to suppress the spread of this respiratory pathogen from one cell to another.

Online Measurement of Real-Time Cytotoxic Responses Induced by Multi-Component Matrices, such as Natural Products, through Electric Cell-Substrate Impedance Sensing (ECIS).

Natural products are complex matrices of compounds that are prone to interfere with the label-dependent methods that are typically used for cytotoxicity screenings. Here, we developed a label-free Electric Cell-substrate Impedance Sensing (ECIS)-based cytotoxicity assay that can be applied in the assessment of the cytotoxicity of natural extracts. The conditions to measure the impedance using ECIS were first optimized in mice immortalized hypothalamic neurons GT1-7 cells. The performance of four natural extracts when tested using three conventional cytotoxicity assays in GT1-7 cells, was studied. Betula pendula (silver birch tree) was found to interfere with all of the cytotoxicity assays in which labels were applied. The silver birch extract was also proven to be cytotoxic and, thus, served as a proof-of-concept for the use of ECIS. The extract was fractionated and the ECIS method permitted the distinction of specific kinetic patterns of cytotoxicity on the fractions as well as the extract's pure constituents. This study offers evidence that ECIS is an excellent tool for real-time monitoring of the cytotoxicity of complex extracts that are difficult to work with using conventional (label-based) assays. Altogether, it offers a very suitable cytotoxicity-screening assay making the work with natural products less challenging within the drug discovery workflow.

New derivatives of dehydroabietic acid target planktonic and biofilm bacteria in Staphylococcus aureus and effectively disrupt bacterial membrane integrity.

The combination of the dehydroabietic acid scaffold with different amino acids resulted in the discovery of a new class of hybrid compounds that targets both planktonic and biofilms bacteria in Staphylococcus aureus strains and are far more potent anti-biofilm agents than conventional antibiotics. Unlike dehydroabietic acid, these compounds can disrupt biofilms within a short time period and compromise the integrity of the bacterial membrane. Two of the compounds identified in our study are the most potent abietane-type anti-biofilm agents reported so far and display robust activity against pre-formed biofilms at concentrations only 3-6-fold higher than those required to inhibit biofilm formation. Their easy preparation based on proteolysis-resistant d- and unusual amino acids makes them useful chemical probes to gain a deeper understanding of bacterial biofilms and outstanding candidates for further development into new drugs to fight infections.

Effective Antibiofilm Polyketides against Staphylococcus aureus from the Pyranonaphthoquinone Biosynthetic Pathways of Streptomyces Species.

Streptomyces bacteria are renowned for their ability to produce bioactive secondary metabolites. Recently, synthetic biology has enabled the production of intermediates and shunt products, which may have altered biological activities compared to the end products of the pathways. Here, we have evaluated the potential of recently isolated alnumycins and other closely related pyranonaphthoquinone (PNQ) polyketides against Staphylococcus aureus biofilms. The antimicrobial potency of the compounds against planktonic cells and biofilms was determined by redox dye-based viability staining, and the antibiofilm efficacy of the compounds was confirmed by viable counting. A novel antistaphylococcal polyketide, alnumycin D, was identified. Unexpectedly, the C-ribosylated pathway shunt product alnumycin D was more active against planktonic and biofilm cells than the pathway end product alnumycin A, where a ribose unit has been converted into a dioxane moiety. The evaluation of the antibiofilm potential of other alnumycins revealed that the presence of the ribose moiety in pyranose form is essential for high activity against preformed biofilms. Furthermore, the antibiofilm potential of other closely related PNQ polyketides was examined. Based on their previously reported activity against planktonic S. aureus cells, granaticin B, kalafungin, and medermycin were also selected for testing, and among them, granaticin B was found to be the most potent against preformed biofilms. The most active antibiofilm PNQs, alnumycin D and granaticin B, share several structural features that may be important for their antibiofilm activity. They are uncharged, glycosylated, and also contain a similar oxygenation pattern of the lateral naphthoquinone ring. These findings highlight the potential of antibiotic biosynthetic pathways as a source of effective antibiofilm compounds.

The Lignan-containing Extract of Schisandra chinensis Berries Inhibits the Growth of Chlamydia pneumonia.

The purpose of this study was to investigate the effect and selectivity of an extract of Schisandra chinensis berries against Chlamydia pneumoniae and C. trachomatis. Among the ethnopharmacological uses of the extract from Schisandrae fructus are cough and pneumonia. Therefore we focused on respiratory pathogens. The extract completely inhibited the growth of C. pneumoniae strain CV6 at 250 µg/mL concentration. The inhibition of C. pneumoniae and C. trachomatis growth was dose dependent and established with three different strains. The extract inhibited C. pneumoniae production of infectious progeny in a dose dependent manner. Chlamydia selectivity was elucidated with growth inhibition measurements of three other respiratory bacterial species. A pure compound found in Schisandra chinensis berries, schisandrin B at 20.0 µg/mL concentration inhibited the growth of both C. pneumoniae and C. trachomatis. The extract was found to be non-toxic to the human host cells. These findings highlight the potential of the extract from Schisandra chinensis berries as a source for antichlamydial compounds.

Inhibitory activity of the isoflavone biochanin A on intracellular bacteria of genus Chlamydia and initial development of a buccal formulation.

Given the established role of Chlamydia spp. as causative agents of both acute and chronic diseases, search for new antimicrobial agents against these intracellular bacteria is required to promote human health. Isoflavones are naturally occurring phytoestrogens, antioxidants and efflux pump inhibitors, but their therapeutic use is limited by poor water-solubility and intense first-pass metabolism. Here, we report on effects of isoflavones against C. pneumoniae and C. trachomatis and describe buccal permeability and initial formulation development for biochanin A. Biochanin A was the most potent Chlamydia growth inhibitor among the studied isoflavones, with an IC50 = 12 µM on C. pneumoniae inclusion counts and 6.5 µM on infectious progeny production, both determined by immunofluorescent staining of infected epithelial cell cultures. Encouraged by the permeation of biochanin A across porcine buccal mucosa without detectable metabolism, oromucosal film formulations were designed and prepared by a solvent casting method. The film formulations showed improved dissolution rate of biochanin A compared to powder or a physical mixture, presumably due to the solubilizing effect of hydrophilic additives and presence of biochanin A in amorphous state. In summary, biochanin A is a potent inhibitor of Chlamydia spp., and the in vitro dissolution results support the use of a buccal formulation to potentially improve its bioavailability in antichlamydial or other pharmaceutical applications.

Recent advances in technologies for developing drugs against Chlamydia pneumoniae.

INTRODUCTION: The unique morphological characteristics, capacity of manipulating host cell function and association with chronic inflammatory diseases represent the features of Chlamydia pneumoniae that have fascinated scientists and medical professionals for several decades. AREAS COVERED: In this paper, the authors review the current status of attempts to discover and develop drugs against C. pneumoniae, including: the discovery of non-conventional antichlamydial agents, targeting chlamydial type 3 secretion system, approved drug repositioning and combination therapies. In addition, the authors discuss the recent advances in C. pneumoniae-related genomics and proteomics research and genetic manipulation technologies. EXPERT OPINION: Based on current knowledge, it is important for researchers to continue to focus on phenotypic assays on persistent infections. There should also be a careful evaluation of the physicochemical properties of the lead candidates and attempts toward more narrow-spectrum antibacterial agents. All these elements are important for successful lead generation. The recent advances in understanding C. pneumoniae biology and breakthroughs in genetic transformation are likely to improve the potential for identifying and validating therapeutic targets within both the bacterium and its host cells.

Staphylococcus aureus biofilm susceptibility to small and potent ß(2,2)-amino acid derivatives.

Small antimicrobial ß(2,2)-amino acid derivatives (Mw < 500 Da) are reported to display high antibacterial activity against suspended Gram-positive strains combined with low hemolytic activity. In the present study, the anti-biofilm activity of six ß(2,2)-amino acid derivatives (A1-A6) against Staphylococcus aureus (ATCC 25923) was investigated. The derivatives displayed IC50 values between 5.4 and 42.8 µM for inhibition of biofilm formation, and concentrations between 22.4 and 38.4 µM had substantial effects on preformed biofilms. The lead derivative A2 showed high killing capacity (log R), and it caused distinct ultrastructural changes in the biofilms as shown by electron and atomic force microscopy. The anti-biofilm properties of A2 was preserved under high salinity conditions. Extended screening showed also high activity of A2 against Escherichia coli (XL1 Blue) biofilms. These advantageous features together with high activity against preformed biofilms make ß(2,2)-amino acid derivatives a promising class of compounds for further development of anti-biofilm agents.

Screening and structural analysis of flavones inhibiting tankyrases.

Flavonoids are known for their beneficial effects on human health, and therefore the therapeutic potential of these compounds have been extensively studied. Flavone has been previously identified as a tankyrase inhibitor, and to further elucidate whether tankyrases would be inhibited by other flavonoids, we performed a systematic screening of tankyrase 2 inhibitory activity using 500 natural and naturally derived flavonoids covering nine different flavonoid classes. All identified tankyrase inhibitors were flavones. We report crystal structures of all the hit compounds in complex with the catalytic domain of human tankyrase 2. Flavone derivatives in all 10 crystal structures bind to the nicotinamide binding site of tankyrase 2. Potencies of the active flavones toward tankyrases vary between 50 nM and 1.1 µM, and flavones show up to 200-fold selectivity for tankyrases over ARTD1. The molecular details of the interactions revealed by cocrystal structures efficiently describe the properties of potent flavone derivatives inhibiting tankyrases.

Evaluation of antibacterial and anti-biofilm activities of cinchona alkaloid derivatives against Staphylococcus aureus.

Bacterial biofilms are resistant to most of the commonly available antibacterial chemotherapies. Thus, an enormous need exists to meet the demands of effective anti-biofilm therapy. In this study, a small library of cinchona alkaloids, including the naturally occurring compounds cinchonidine and cinchonine, as well as various synthetic derivatives and analogues was screened for antibacterial and anti-biofilm activity against the Staphylococcus aureus biofilm producing strain ATCC 25923. Two methods were used to evaluate activity against biofilms, namely crystal violet staining to measure biomass and resazurin assay to measure biofilms viability. Cinchonidine was found to be inactive, whereas a synthetic derivative, 11-triphenylsilyl-10,11-dihydrocinchonidine (11-TPSCD), was effective against planktonic bacteria as well as in preventing biofilm formation at low micromolar concentrations. Higher concentrations were required to eradicate mature biofilms.

Corn mint (Mentha arvensis) extract diminishes acute Chlamydia pneumoniae infection in vitro and in vivo.

Corn mint ( Mentha arvensis ) provides a good source of natural phenols such as flavone glycosides and caffeic acid derivatives, which may have prophylactic properties against inflammations. This study investigated whether corn mint extract would be beneficial against a universal respiratory tract pathogen, Chlamydia pneumoniae , infection. The extract inhibited the growth of C. pneumoniae CWL-029 in vitro in a dose-dependent manner. The inhibition was confirmed against a clinical isolate K7. The phenolic composition of the extract was analyzed by UPLC-ESI/Q-TOF/MS, the main components being linarin and rosmarinic acid. These compounds were active in vitro against C. pneumoniae. Linarin completely inhibited the growth at 100 µM. Inbred C57BL/6J mice were inoculated with C. pneumoniae K7. M. arvensis extract was given intraperitoneally once daily for 3 days prior to inoculation and continued for 10 days postinfection. The extract was able to diminish the inflammatory parameters related to C. pneumoniae infection and significantly (p = 0.019) lowered the number of C. pneumoniae genome equivalents detected by PCR at biologically relevant amounts.

Effects of coadministration of natural polyphenols with doxycycline or calcium modulators on acute Chlamydia pneumoniae infection in vitro.

Besides small molecules from medicinal chemistry, natural products are still major sources of innovative therapeutic agents for various conditions, including infectious diseases. Here we present the first attempt to design a combination treatment targeted against Chlamydia pneumoniae infection using coadministration of natural phenolics with calcium (Ca(2+)) modulators, and also the concomitant administration of these compounds with doxycycline. An in vitro acute C. pneumoniae model in human lung epithelial cells was used and Loewe additivity model was applied to evaluate the effects. In general, the phenolic compounds, quercetin, luteolin, rhamnetin and octyl gallate did not improve the antichlamydial effect of doxycycline, and, in some cases, resulted in antagonistic effects. The combination of doxycycline and Ca(2+) modulators (isradipine, verapamil and thapsigargin) was at most additive, and at subinhibitory concentrations of doxycycline, often even antagonistic. The Ca(2+) modulators showed no inhibitory effects on C. pneumoniae growth alone, whereas the coadminstration of Ca(2+) modulators with phenolic compounds resulted in potentiation of the antichlamydial effect of phenolic compounds. Verapamil (100 µM) was synergistic with low quercetin and luteolin concentrations (0.39 and 1.56 µM), whereas 10 µM isradipine was synergistic with high quercetin, rhamnetin and octyl gallate concentrations (12.5 µM and 100 µM). Use of thapsigargin with the phenolic compounds resulted in the most intense synergism. Interaction indices 0.12 and 0.14 were achieved with 0.39 µM luteolin and 10 and 100 nM thapsigargin, respectively. To conclude, the observed results indicate that the Ca(2+) modulators potentiate the antichlamydial effects of the phenolic compounds.

Discovery of dual binding site acetylcholinesterase inhibitors identified by pharmacophore modeling and sequential virtual screening techniques.

Dual binding site acetylcholinesterase (AChE) inhibitors are promising for the treatment of Alzheimer's disease (AD). They alleviate the cognitive deficits and AD-modifying agents, by inhibiting the ß-amyloid (Aß) peptide aggregation, through binding to both the catalytic and peripheral anionic sites, the so called dual binding site of the AChE enzyme. In this Letter, chemical features based 3D-pharmacophore models were developed based on the eight potent and structurally diverse AChE inhibitors (I-VIII) obtained from high-throughput in vitro screening technique. The best 3D-pharmacophore model, Hypo1, consists of two hydrogen-bond acceptor lipid, one hydrophobe, and two hydrophobic aliphatic features obtained by Catalyst/HIPHOP algorithm adopted in Discovery studio program. Hypo1 was used as a 3D query in sequential virtual screening study to filter three small compound databases. Further, a total of nine compounds were selected and followed on in vitro analysis. Finally, we identified two leads--Specs1 (IC(50)=3.279 µM) and Spec2 (IC(50)=5.986 µM) dual binding site compounds from Specs database, having good AChE enzyme inhibitory activity.

Polyketide derivatives active against Botrytis cinerea in Gerbera hybrida.

A previously isolated cDNA molecule from Gerbera hybrida (Asteraceae) codes for a new chalcone synthase-like polyketide synthase, 2-pyrone synthase (2PS). 2PS is able to synthesise 4-hydroxy-6-methyl-2-pyrone (triacetolactone), a putative precursor for gerberin and parasorboside, two abundant glucosides in gerbera. In this study, we show that gerbera plants transformed with the gene for 2PS in an antisense orientation and unable to synthesise gerberin and parasorboside are susceptible to Botrytis cinerea infection. In addition to the preformed glucosides, the transgenic plants also lack several compounds that are induced in control plants when infected with the mould. Some of these induced substances are effective in inhibiting fungal growth both in vitro and in vivo. Two of the phytoalexins were identified as the aglycones of gerberin and trans-parasorboside. The third phytoalexin is a rare coumarin, 4-hydroxy-5-methylcoumarin; however, it is typical of many plants of the sunflower family Asteraceae. The coumarin cannot be structurally derived from either gerberin or parasorboside, but may be derived from a related polyketide intermediate.

Molecular Docking Guided Comparative GFA, G/PLS, SVM and ANN Models of Structurally Diverse Dual Binding Site Acetylcholinesterase Inhibitors.

Recently discovered 42 AChE inhibitors binding at the catalytic and peripheral anionic site were identified on the basis of molecular docking approach, and its comparative quantitative structure-activity relationship (QSAR) models were developed. These structurally diverse inhibitors were obtained by our previously reported high-throughput in vitro screening technique using 384-well plate's assay based on colorimetric method of Ellman. QSAR models were developed using (i) genetic function algorithm, (ii) genetic partial least squares, (iii) support vector machine and (iv) artificial neural network techniques. The QSAR model robustness and significance was critically assessed using different cross-validation techniques on test data set. The generated QSAR models using thermodynamic, electrotopological and electronic descriptors showed that nonlinear methods are more robust than linear methods, and provide insight into the structural features of compounds that are important for AChE inhibition.

Miniaturization and validation of the Ellman's reaction based acetylcholinesterase inhibitory assay into 384-well plate format and screening of a chemical library.

The aim of this study was to screen for acetylcholinesterase (AChE) inhibitors from a large chemical library of commercially available compounds. For this purpose, the Ellman's reaction based assay was miniaturized into 384-well plate format, and two modifications of the kinetic protocol were studied with the aim of developing a rapid screening platform that could ensure high efficiency in finding true hits. It was proven that when starting the kinetic reaction by addition of the substrate, better assay performance was achieved and more practical benefits obtained. Using the optimized automated protocol, a chemical library of 56,320 compounds was screened. A total of 350 positive hits were identified and their IC50 calculated. Three highly active compounds were identified with IC50 values close or even lower to physostigmine (< 0.1 microM). The activity towards butyrylcholinesterase (BChE) of these three most active hits was also evaluated. The most active hit (IC(50(AChE)) = 0.019 microM), was identified as a new inhibitor, belonging to ChemDiv chemical library: (N-[3-(3,5-dimethyl-1-piperidinyl)propyl]-5-ethyl-2-methyl-8-oxo-thieno[2',3':4,5]pyrrolo[1,2-d] [1,2,4] triazine-7(8H)-acetamid), with no other biological activities reported until now. The interactions of this hit with both cholinesterases were further analyzed using computational docking studies. To our knowledge, this is the largest published screening campaign of commercially available compounds that has focused on finding new AChE inhibitors. The miniaturized 384-well plate format of the Ellman's method was proven to be robust and to perform reliably.

The binding of synthetic retinoids to lipocalin beta-lactoglobulins.

The binding of therapeutically relevant synthetic retinoid derivatives to bovine and reindeer beta-lactoglobulin (betaLG) is demonstrated using fluorescence quenching and ultrafiltration/HPLC methods. Furthermore, synthesis of methyl (E)-3-[4-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)vinyl]phenyl]-acrylate 4 and (E)-3-[4-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)vinyl]phenyl]acrylic acid 5 is described. All studied compounds bind to both betaLG homologues with nanomolar K(d) values, and the interaction diminishes the pH-dependent aggregation of retinoids. Thus, betaLG may show benefits in improving the bioavailability of retinoid derivatives.