Adyary Fallarero

Towards fabrication of 3D printed medical devices to prevent biofilm formation

The use of three-dimensional (3D) printing technologies is transforming the way that materials are turned into functional devices. We demonstrate in the current study the incorporation of anti-microbial nitrofurantoin in a polymer carrier material and subsequent 3D printing of a model structure, which resulted in an inhibition of biofilm colonization. The approach taken is very promising and can open up new avenues to manufacture functional medical devices in the future.

Inhibition of acetylcholinesterase by coumarins: The case of coumarin 106

In this contribution, from a coumarin library consisting of 29 compounds including natural and synthetic derivatives, an active acetylcholinesterase (AChE) inhibitor (coumarin 106) was found. This circumstance leaded us to continue with the pharmacological characterization of coumarin 106. The first study with the coumarin library was performed using a 96-microtiter well plate assay based on Ellmans reaction. Coumarins were assayed at 5 and 30 microM, and coumarin 106 was found the most active inhibitor at both concentrations. The follow-up analysis using kinetic studies demonstrated that coumarin 106 displays mixed-type AChE inhibition with a pIC(50)=4.97+/-0.09 and K(i)=2.36+/-0.17 microM. The ability of this molecule to interact with AChE was further confirmed through computational studies, in which a primary binding was proved to occur at the active gorge site, while a secondary binding was demonstrated at the peripheral anionic site. Also, coumarin 106 was shown to inhibit butyrylcholinesterase (BChE) with slightly lower potency (pIC(50)=4.56+/-0.06), and found to be non-toxic in Caco-2 cells. The combination of these findings makes coumarin 106 an attractive molecule for further investigation. This is the first report where AChE inhibitory activity has been associated with coumarin 106, and proof has been given of its convenience as a lead molecule.

Pros and cons of using resazurin staining for quantification of viable Staphylococcus aureus biofilms in a screening assay

Staining of Staphylococcus aureus biofilms with 20 microM resazurin during 20 min was shown to provide a good screening assay in 96-well micro titer plates. However, data quality was found to be dependent on the staining duration and biofilm concentration. Also, the inadequacy of using resazurin calibration curves with planktonic cells to estimate S. aureus biofilm concentrations was demonstrated.

Automating a 96-well microtitre plate model for Staphylococcus aureus biofilms : an approach to screening of natural antimicrobial compounds

The purpose of this study was to establish and automate an assay to be used for screening novel antimicrobial agents against biofilm-forming Staphylococcus aureus bacteria. The selected assay was based on crystal violet staining, which is a well used method for staining bacterial biofilms. The method was first optimised manually, antibiotic susceptibility was established and biofilm formation in plates was confirmed using atomic force microscopy. Automation of the assay was done using a Thermo Scientific Multidrop((R)) Combi dispenser and Biomek((R)) 3000 liquid handling workstation. A detailed comparison of the performance between the manual and the automated method was made in terms of screening window coefficient as well as other statistical parameters and repeatability measurements, such as plate-to-plate and day-to-day variability. Automated screening of an in-house library of natural products gave the same positive hits as previously reported, therefore the developed assay can be regarded as a reliable screening tool.

Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms

When single-cell (or suspended) bacteria switch into the biofilm lifestyle, they become less susceptible to antimicrobials, imposing the need for anti-biofilms research. Flavonoids are among the most extensively studied natural compounds with an unprecedented amount of bioactivity claims. Most studies focus on the antibacterial effects against suspended cells; fewer reports have researched their anti-biofilm properties. Here, a high throughput phenotypic platform was utilized to screen for the inhibitory activity of 500 flavonoids, including natural and synthetic derivatives, against Staphylococcus aureus biofilms. Since discrepancies among results from earlier antibacterial studies on flavonoids had been noted, the current study aimed to minimize sources of variations. After the first screen, flavonoids were classified as inactive (443), moderately active (47) or highly active (10). Further, exclusion criteria combining bioactivity and selectivity identified two synthetic flavans as the most promising. The body of data reported here serves three main purposes. First, it offers an improved methodological workflow for anti-biofilm screens of chemical libraries taking into account the (many times ignored) connections between anti-biofilm and antibacterial properties. This is particularly relevant for the study of flavonoids and other natural products. Second, it provides a large and freely available anti-biofilm bioactivity dataset that expands the knowledge on flavonoids and paves the way for future structure-activity relationship studies and structural optimizations. Finally, it identifies two new flavans that can successfully act on biofilms, as well as on suspended bacteria and represent more feasible antibacterial candidates.

Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

Despite that three types of assays (measuring biofilm viability, biomass, or matrix) are described to assess anti-biofilm activity, they are rarely used together. As infections can easily reappear if the matrix is not affected after antibiotic treatments, our goal was to explore the simultaneous effects of antibiotics on the viability, biomass and matrix of Staphylococcus aureus biofilms (ATCC 25923). Viability and biomass were quantified using resazurin and crystal violet staining sequentially in the same plate, while matrix staining was conducted with a wheat germ agglutinin-Alexa Fluor 488 fluorescent conjugate. Establishment of the detection limits and linearity ranges allowed concluding that all three methods were able to estimate biofilm formation in a similar fashion. In a susceptibility study with 18-h biofilms, two model compounds (penicillin G and ciprofloxacin) caused a reduction on the viability and biomass accompanied by an increase or not changed levels of the matrix, respectively. This response pattern was also proven for S. aureus Newman, S. epidermidis and E. coli biofilms. A classification of antibiotics based on five categories according to their effects on viability and matrix has been proposed earlier. Our data suggests a sixth group, represented by penicillin, causing decrease in bacterial viability but showing stimulatory effects on the matrix. Further, if effects on the matrix are not taken into account, the long-term chemotherapeutic effect of antibiotics can be jeopardized in spite of the positive effects on biofilms viability and biomass. Thus, measuring all these three endpoints simultaneously provide a more complete and accurate picture.

Interaction of (benzylidene-hydrazono)-1,4-dihydropyridines with β-amyloid, acetylcholine, and butyrylcholine esterases

Approved drugs for the treatment of Alzheimers disease belong to the group of inhibitors of the acetylcholinesterase (AChE) and NMDA receptor inhibitors. However none of the drugs is able to combat or reverse the progression of the disease. Thus, the recently reported promising multitarget-directed molecule approach was applied here. Using the lead compound DUO3, which was found to be a potent inhibitor of the AChE and butyrylcholinesterase (BuChE) as well as an inhibitor of the formation of the amyloid (Abeta) plaque, new non-permanently positively charged derivatives were synthesized and biologically characterized. In contrast to DUO3 the new bisphenyl-substituted pyridinylidene hydrazones 5 are appropriate to cross the blood-brain barrier due to their pK(a) values and lipophilicity, and to inhibit both the AChE and BuChE. More important some of the pyridinylidene hydrazones inhibit the Abeta fibril formation completely and destruct the already formed fibrils significantly.

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.

Exploration of Natural Compounds as Sources of New Bifunctional Scaffolds Targeting Cholinesterases and Beta Amyloid Aggregation: The Case of Chelerythrine

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the ADs pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin-chelerythrine-acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.

Alpha- and β-casein components of host milk induce biofilm formation in the mastitis bacterium Streptococcus uberis.

Streptococcus uberis is an environmental udder pathogen that infects cattle and can cause persistent intramammary infection (IMI), despite the fact that isolates are mainly susceptible to antibiotics. As biofilm growth can cause persistent infection, the ability of ten S. uberis isolates from clinical and subclinical IMIs to form biofilms on the polystyrene surface of a conventional 96-microplates model was examined. Biofilm formation was judged by different staining methods (crystal violet and resazurin) and by atomic force and fluorescence microscopy. These analyses revealed that two out of ten S. uberis strains tested were able to form biofilms. Upon treatment with Proteinase K, biofilms of S. uberis were completely disintegrated, which indicates that biofilm formation is protein-mediated in these strains. Addition of trace amounts of milk, the natural growth medium of S. uberis, significantly increased biofilm formation by most of the strains initially classified as non-biofilm producers. Alpha-casein and β-casein were the primary inducers of biofilm growth, and casein degradation by serine protease activity was required to achieve maximal biofilm production. These results suggest that the extracellular proteolytic activity of S. uberis contributes to an increased biofilm formation. Such a mode of growth induced by host proteins might help to explain the persistence of IMIs caused by this pathogen.