Quantification of Enzymatic Biofilm Removal Using the Sauerbrey Equation: Application to the Case of Pseudomonas protegens.

A methodology for the quantitative analysis of enzymatic removal of biofilms (BF) was developed, based on a quartz crystal microbalance (QCM) under stationary conditions. This was applied to the case of Pseudomonas protegens (PP) BFs, through a series of five enzymes, whose removal activity was screened using the presented methodology. The procedure is based on the following: when BFs can be modeled as rigid materials, QCM can be used as a balance under stationary conditions for determining the BFs mass reduction by enzymatic removal. For considering a BF as a rigid model, energy dissipation effects, associated with viscoelastic properties of the BF, must be negligible. Hence, a QCM system with detection of dissipation (referred to as QCM with dissipation) was used for evaluating the energy losses, which, in fact, resulted in negligible energy losses in the case of dehydrated PP BFs, validating the application of the Sauerbrey equation for the change of mass calculations. The stationary methodology reduces operating times and simplifies data analysis in comparison to dynamic approaches based on flow setups, which requires the incorporation of dissipation effects due to the liquid media. By carrying out QCM, glycosidase-type enzymes showed BF removal higher than 80% at enzyme concentration 50 ppm, reaching removal over 90% in the cases of amylase and cellulase/xylanase enzymes. The highest removal percentage produced a reduction from about 15 to 1 µg in the BF mass. Amylase enzyme was tested from below 50 to 1 ppm, reaching around 60% of removal at 1 ppm. The obtained results were supported by other instrumental techniques such as Raman spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, atomic force microscopy, high performance anion exchange chromatography, thermogravimetric analysis, and differential scanning calorimetry. The removal quantifications obtained with QCM were compared with those obtained by well-established screening techniques (UV-vis spectrophotometry using crystal violet and agar diffusion test). The proposed methodology expands the possibility of using a quartz microbalance to perform enzymatic activity screening.

Synthesis of Oligosaccharides Containing the S-Galp(α1 → 3)Galp Unit, Glycomimetic of the Epitope Recognized by Lytic Antibodies.

Two important activities take place in the surface of Trypanosoma cruzi, the agent of Chagas disease: the trans-sialidase (TcTS) catalyzes the transfer of sialic acid from the host glycoconjugates to the mucin-like glycoproteins from the parasite and the presence of lytic antibodies recognize the epitope α-Galp(1 → 3)-ß-Galp(1 → 4)-α-GlcNAcp. This antigenic structure is known to be present in the parasite mucins; however, in order to be substrates of trans-sialidase, some of the galactose residues should be in the ß-Galp configuration. To study the interaction between both activities, it is important to count the synthetic structures as well as the structural-related glycomimetics. With this purpose, we addressed the synthesis of a trisaccharide and two isomeric tetrasaccharides containing the 1-S-α-Galp(1 → 3)-ß-Galp motif, the thio analog of the epitope recognized by lytic antibodies. Starting with a common lactose precursor, the sulfur function was incorporated by double inversion of the configuration of the galactose residue that was further glycosylated using different activated donors. Both tetrasaccharides were good acceptors of sialic acid in the reaction catalyzed by TcTS, as determined by high-performance anion exchange chromatography.

trans-Sialylation: a strategy used to incorporate sialic acid into oligosaccharides.

Sialic acid, as a component of cell surface glycoconjugates, plays a crucial role in recognition events. Efficient synthetic methods are necessary for the supply of sialosides in enough quantities for biochemical and immunological studies. Enzymatic glycosylations obviate the steps of protection and deprotection of the constituent monosaccharides required in a chemical synthesis. Sialyl transferases with CMP-Neu5Ac as an activated donor were used for the construction of α2-3 or α2-6 linkages to terminal galactose or N-acetylgalactosamine units. trans-Sialidases may transfer sialic acid from a sialyl glycoside to a suitable acceptor and specifically construct a Siaα2-3Galp linkage. The trans-sialidase of Trypanosoma cruzi (TcTS), which fulfills an important role in the pathogenicity of the parasite, is the most studied one. The recombinant enzyme was used for the sialylation of ß-galactosyl oligosaccharides. One of the main advantages of trans-sialylation is that it circumvents the use of the high energy nucleotide. Easily available glycoproteins with a high content of sialic acid such as fetuin and bovine κ-casein-derived glycomacropeptide (GMP) have been used as donor substrates. Here we review the trans-sialidase from various microorganisms and describe their application for the synthesis of sialooligosaccharides.

Structure-Based Virtual Screening of New Benzoic Acid Derivatives as Trypanosoma cruzi Trans-sialidase Inhibitors.

BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, represents a worldwide epidemiological, economic, and social problem. In the last decades, the trans-sialidase enzyme of Trypanosoma cruzi has been considered an attractive target for the development of new agents with potential trypanocidal activity. OBJECTIVE: In this work, the aim was to find new potential non-sugar trans-sialidase inhibitors using benzoic acid as a scaffold. METHODS: A structure-based virtual screening of the ZINC15 database was carried out. Additionally, the enzyme and trypanocidal activity of the selected compounds was determined. RESULTS: The results of this work detected 487 compounds derived from benzoic acid as potential transsialidase inhibitors with a more promising binding energy value (< -7.7 kcal/mol) than the known inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA). In particular, two lead compounds, V1 and V2, turned out to be promising trans-sialidase inhibitors. Even though the trypanocidal activity displayed was low, these compounds showed trans-sialidase inhibition values of 87.6% and 29.6%, respectively. CONCLUSION: Structure-based virtual screening using a molecular docking approach is a useful method for the identification of new trans-sialidase inhibitors.

Trypanosoma cruzi trans-sialidase. A tool for the synthesis of sialylated oligosaccharides.

Cells are covered by a complex array of carbohydrates. Among them, sialosides are of key importance in intracellular adhesion, recognition and signaling. The need for structurally diverse sialosides impelled the search for efficient synthetic methods since their isolation from natural sources is a difficult task. The enzymatic approach obviates the need of a chemical synthesis for protecting or participating groups in the substrates. The trans-sialidase of Trypanosoma cruzi (TcTS) is highly stereospecific for the transfer of sialic acid from an α-sialylglycoside donor to a terminal ß-galactopyranosyl unit in the acceptor substrate to form the α-Neu5Ac-(2 → 3)-ß-D-Galp motif. The enzyme was cloned and easily available glycoproteins, e.g. fetuin, may be used as donors of sialic acid, constituting strong points for the scalability of TcTS-catalyzed reactions. This review outlines the preparative use of TcTS for the sialylation of oligosaccharides. A detailed description of the substrates used as sialic acid donors, the acceptor substrates and the methods employed to monitor the reaction is included.

Trypanosoma cruzi trans-sialidase alternative substrates: Study of the effect of substitution in C-6 in benzyl ß-lactoside.

Trypanosoma cruzi trans-sialidase (TcTS) is a cell surface protein that participates in the adhesion and invasion mechanisms of the parasite into the host cells, making it an attractive target for inhibitors design. In order to contribute to the knowledge of the interaction between TcTS and their acceptor substrates, we designed and synthesized a library of 20 benzyl lactosides substituted in C-6 of the glucose residue with a series of 1,2,3-triazole derivatives containing different aromatic substituents in the C-4 position. The library was prepared by alkyne-azide cycloaddition reaction catalyzed by Cu(I) ("click chemistry") between a benzyl ß-lactoside functionalized with an azide group in the C-6 position and a series of 2-propargyl phenyl ethers. Herein we analyzed the chromatographic behavior on high performance anion exchange chromatography (HPAEC) of the triazoyl-lactose derivatives and their activity as acceptors of TcTS and inhibitors of the sialylation of N-acetyllactosamine. The triazoyl derivatives were obtained with excellent yields and all of them behaved as moderate alternative substrates. The presence of bulky hydrophobic substituents dramatically increased the retention times in HPAEC but did not affect significantly their acceptor properties toward TcTS.

Synthesis, molecular docking and biological evaluation of novel phthaloyl derivatives of 3-amino-3-aryl propionic acids as inhibitors of Trypanosoma cruzi trans-sialidase.

In the last two decades, trans-sialidase of Trypanosoma cruzi (TcTS) has been an important pharmacological target for developing new anti-Chagas agents. In a continuous effort to discover new potential TcTS inhibitors, 3-amino-3-arylpropionic acid derivatives (series A) and novel phthaloyl derivatives (series B, C and D) were synthesized and molecular docking, TcTS enzyme inhibition and determination of trypanocidal activity were carried out. From four series obtained, compound D-11 had the highest binding affinity value (-11.1 kcal/mol) compared to reference DANA (-7.8 kcal/mol), a natural ligand for TS enzyme. Furthermore, the 3D and 2D interactions analysis of compound D-11 showed a hydrogen bond, π-π stacking, π-anion, hydrophobic and Van der Waals forces with all important amino acid residues (Arg35, Arg245, Arg314, Tyr119, Trp312, Tyr342, Glu230 and Asp59) on the active site of TcTS. Additionally, D-11 showed the highest TcTS enzyme inhibition (86.9% ±â€¯5) by high-performance ion exchange chromatography (HPAEC). Finally, D-11 showed better trypanocidal activity than the reference drugs nifurtimox and benznidazole with an equal % lysis (63 ±â€¯4 and 65 ±â€¯2 at 10 µg/mL) and LC50 value (52.70 ±â€¯2.70 µM and 46.19 ±â€¯2.36 µM) on NINOA and INC-5 strains, respectively. Therefore, D-11 is a small-molecule with potent TcTS inhibition and a strong trypanocidal effect that could help in the development of new anti-Chagas agents.

Synthesis of a model trisaccharide for studying the interplay between the anti α-Gal antibody and the trans-sialidase reactions in Trypanosoma cruzi.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense glycocalix mainly composed by glycoproteins called mucins which are also the acceptors of sialic acid in a reaction catalyzed by a trans-sialidase (TcTS). Sialylation of trypomastigote mucins protects the parasite from lysis by the anti α-Galp antibodies from serum. The TcTS is essential for the infection process since T. cruzi is unable to biosynthesize sialic acid. The enzyme specifically transfers it from a terminal ß-d-Galp unit in the host glycoconjugate to terminal ß-d-Galp units in the parasite mucins to construct the d-NeuNAc(α2→3)ß-d-Galp motif. On the other hand, although galactose is the most abundant sugar in mucins of both, the infective trypomastigotes and the insect stage epimastigotes, α-d-Galp is only present in the infective stage whereas ß-d-Galf is characteristic of the epimastigote stage of the less virulent strains. Neither α-d-Galp nor d-Galf is acceptor of sialic acid. In the mucins, some of the oligosaccharides are branched with terminal ß-d-Galp units to be able to accept sialic acid in the TcTS reaction. Based on previous reports showing that anti α-Galp antibodies only partially colocalize with sialic acid, we have undertaken the synthesis of the trisaccharide α-d-Galp(1→3)-[ß-d-Galp(1→6)]-d-Galp, the smallest structure containing both, the antigenic d-Galp(α1→3)-d-Galp unit and the sialic acid-acceptor ß-d-Galp unit. The trisaccharide was obtained as the 6-aminohexyl glycoside to facilitate further conjugation for biochemical studies. The synthetic approach involved the α-galactosylation at O-4 of a suitable precursor of the reducing end, followed by ß-galactosylation at O-6 of the same precursor and introduction of the 6-aminohexyl aglycone. The fully deprotected trisaccharide was successfully sialylated by TcTS using either 3'-sialyllactose or fetuin as donors. The product, 6-aminohexyl α-d-NeuNAc(2→3)-ß-d-Galp(1→6)-[α-d-Galp(1→3)]-ß-d-Galp, was purified and characterized.

An in vitro and in vivo evaluation of new potential trans-sialidase inhibitors of Trypanosoma cruzi predicted by a computational drug repositioning method.

Chagas disease is one of the most important neglected parasitic diseases afflicting developed and undeveloped countries. There are currently limited options for inexpensive and secure pharmacological treatment. In this study, we employed a structure-based virtual screening protocol for 3180 FDA-approved drugs for repositioning of them as potential trans-sialidase inhibitors. In vitro and in vivo evaluations were performed for the selected drugs against trypomastigotes from the INC-5 and NINOA strains of T. cruzi. Also, inhibition of sialylation by the trans-sialidase enzyme reaction was evaluated using high-performance anion-exchange chromatography with pulse amperometric detection to confirm the mechanism of action. Results from the computational study showed 38 top drugs with the best binding-energies. Four compounds with antihistaminic, anti-hypertensive, and antibiotic properties showed better trypanocidal effects (LC50 range = 4.5-25.8 µg/mL) than the reference drugs, nifurtimox and benznidazole (LC50 range = 36.1-46.8 µg/mL) in both strains in the in vitro model. The anti-inflammatory, sulfasalazine showed moderate inhibition (37.6%) of sialylation in a trans-sialidase enzyme inhibition reaction. Sulfasalazine also showed the best trypanocidal effects in short-term in vivo experiments on infected mice. This study suggests for the first time that the anti-inflammatory sulfasalazine could be used as a lead compound to develop new trans-sialidase inhibitors.

Multivalent sialylation of ß-thio-glycoclusters by Trypanosoma cruzi trans sialidase and analysis by high performance anion exchange chromatography.

The synthesis of multivalent sialylated glycoclusters is herein addressed by a chemoenzymatic approach using the trans-sialidase of Trypanosoma cruzi (TcTS). Multivalent ß-thio-galactopyranosides and ß-thio-lactosides were used as acceptor substrates and 3'-sialyllactose as the sialic acid donor. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was shown to be an excellent technique for the analysis of the reaction products. Different eluting conditions were optimized to allow the simultaneous resolution of the sialylated species, as well as their neutral precursors. The TcTS efficiently transferred sialyl residues to di, tri, tetra and octa ß-thiogalactosides. In the case of an octavalent thiolactoside, up to six polysialylated compounds could be resolved. Preparative sialylation reactions were performed using the tetravalent and octavalent acceptor substrates. The main sialylated derivatives could be unequivocally assigned by MALDI mass spectrometry. Inhibition of the transfer to the natural substrate, N-acetyllactosamine, was also studied. The octalactoside caused 82 % inhibition of sialic acid transfer when we used equimolar concentrations of donor, acceptor and inhibitor.