RESUMO
Current treatments available for African sleeping sickness or human African trypanosomiasis (HAT) are limited, with poor efficacy and unacceptable safety profiles. Here, we report a new approach to address treatment of this disease based on the use of compounds that bind to parasite surface glycans leading to rapid killing of trypanosomes. Pradimicin and its derivatives are non-peptidic carbohydrate-binding agents that adhere to the carbohydrate moiety of the parasite surface glycoproteins inducing parasite lysis in vitro. Notably, pradimicin S has good pharmaceutical properties and enables cure of an acute form of the disease in mice. By inducing resistance in vitro we have established that the composition of the sugars attached to the variant surface glycoproteins are critical to the mode of action of pradimicins and play an important role in infectivity. The compounds identified represent a novel approach to develop drugs to treat HAT.
RESUMO
BACKGROUND: Many treatment options especially for cancer show a low efficacy for the majority of patients demanding improved biomarker panels for patient stratification. Changes in glycosylation are a hallmark of many cancers and inflammatory diseases and show great potential as clinical disease markers. The large inter-subject variability in glycosylation due to hereditary and environmental factors can complicate rapid transfer of glycan markers into the clinical practice but also presents an opportunity for personalized medicine. SCOPE OF REVIEW: This review discusses opportunities of glycan biomarkers in personalized medicine and reviews the methodology for N-glycan analysis with a specific focus on methods for absolute quantification. MAJOR CONCLUSIONS: The entry into the clinical practice of glycan markers is delayed in large part due to a lack of adequate methodology for the precise and robust quantification of protein glycosylation. Only absolute glycan quantification can provide a complete picture of the disease related changes and will provide the method robustness required by clinical applications. GENERAL SIGNIFICANCE: Glycan biomarkers have a huge potential as disease markers for personalized medicine. The use of stable isotope labeled glycans as internal standards and heavy-isotope labeling methods will provide the necessary method precision and robustness acceptable for clinical use. This article is part of a Special Issue entitled "Glycans in personalized medicine" Guest Editor: Professor Gordan Lauc.
Assuntos
Glicômica/métodos , Polissacarídeos , Medicina de Precisão/métodos , Biomarcadores , Humanos , Polissacarídeos/genética , Polissacarídeos/metabolismoRESUMO
Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of (13)C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was (13)C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.
Assuntos
Espectrometria de Massas/métodos , Polissacarídeos/análise , Polissacarídeos/biossíntese , Isótopos de Carbono , Polissacarídeos/síntese química , Polissacarídeos/química , Padrões de ReferênciaRESUMO
The adsorption of proteins on surfaces often results in a change of their structural behavior and consequently, a loss of bioactivity. One experimental method to study interactions on a molecular level is single molecular force spectroscopy that permits to measure forces down to the pico-newton range. In this work, the binding force between human serum albumin (HSA), covalently immobilized on glutaraldehyde modified gold substrates, and ibuprofen sodium salt was studied by means of single molecular force spectroscopy. First of all, a protocol was established to functionalize atomic force microscopy (AFM) tips with ibuprofen. The immobilization protocol was additionally tested by quartz crystal microbalance with dissipation (QCM-D) and contact angle measurements. AFM was used to characterize the adsorption of HSA on gold substrates, which lead to a packed monolayer of thickness slightly lower than the reported value in solution. Finally, single molecule spectroscopy results were used to characterize the binding force between albumin and ibuprofen and calculate the distance of the transition state (0.6 nm) and the dissociation rate constant (0.055 s(-1)). The results might indicate that part of the adsorbed protein still preserves its functionality upon adsorption.
Assuntos
Ibuprofeno/química , Microscopia de Força Atômica/métodos , Técnicas de Microbalança de Cristal de Quartzo , Albumina Sérica/química , HumanosRESUMO
Weathering steel has been employed for the first time to prepare sample plates for matrix-free laser desorption ionisation mass spectrometry (LDI-MS) of small molecules up to a mass range of around 1500 Da. The effective UV absorption, heat conductivity and porosity of the nanostructured inner rust layer formed during passivation determine the excellent performance in LDI-MS for a broad range of different analyte classes. The inexpensive material was evaluated in a series of relevant analytical applications ranging from the matrix-free detection of serum metabolites, lactose quantification, lipid analysis in milk to the glycoprofiling of antibodies and imaging mass spectrometry of brain tissue samples.
Assuntos
Nanoestruturas , Preparações Farmacêuticas/análise , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aço , Humanos , Preparações Farmacêuticas/sangue , Polissacarídeos/sangue , Espectrofotometria UltravioletaRESUMO
Glycan arrays have become the premier tool for rapidly establishing the binding or substrate specificities of lectins and carbohydrate-processing enzymes. New approaches for accelerating carbohydrate synthesis to address the enormous complexity of natural glycan structures are necessary. Moreover, optimising glycan immobilisation is key for the development of selective, sensitive and reproducible array-based assays. We present a tag-based approach that accelerates the preparation of glycan arrays on all levels by improving the synthesis, the purification and immobilisation of oligosaccharides. Glycan primers were chemically attached to bifunctional polyethyleneglycol (PEG) tags, extended enzymatically with the help of recombinant glycosyltransferases and finally purified by ultrafiltration. When printed directly onto activated glass slides, these glycoPEG tags afforded arrays with exceptionally high sensitivity, low background and excellent spot morphology. Likewise, the conjugation of glycoPEG tags to latex nanoparticles yielded multivalent scaffolds for carbohydrate-binding assays with very low non-specific binding.
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Glicosiltransferases/metabolismo , Lectinas/química , Polietilenoglicóis/síntese química , Polissacarídeos/síntese química , Glicosiltransferases/genética , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismoRESUMO
By combining electrophoretic protein separation with lectin-array-based glycan profiling into a single experiment, we have developed a high-throughput method for the rapid analysis of protein glycosylation in biofluids. Fluorescently tagged proteins are separated by SDS-PAGE and transferred by diffusion to a microscope slide covered with multiple copies of 20 different lectins, where they are trapped by specific carbohydrate protein interactions while retaining their relative locations on the gel. A fluorescence scan of the slide then provides an affinity profile with each of the 20 lectins containing a wealth of structural information regarding the present glycans. The affinity of the employed lectins toward N-glycans was verified on a glycan array of 76 structures. While current lectin-based methods for glycan analysis provide only a picture of the bulk glycosylation in complex protein mixtures or are focused on a few specific known biomarkers, our array-based glycoproteomics method can be used as a biomarker discovery tool for the qualitative exploration of protein glycosylation in an unbiased fashion.
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Glicosilação , Lectinas/química , Análise Serial de Proteínas/métodos , Carboidratos/química , Misturas Complexas , Eletroforese em Gel de Poliacrilamida , Epitopos , Ensaios de Triagem em Larga Escala , Análise em Microsséries , Polissacarídeos/química , Proteômica , Espectrometria de FluorescênciaRESUMO
An effective chemoenzymatic strategy is reported that has allowed the construction, for the first time, of a focused microarray of synthetic N-glycans. Based on modular approaches, a variety of N-glycan core structures have been chemically synthesized and covalently immobilized on a glass surface. The printed structures were then enzymatically diversified by the action of three different glycosyltransferases in nanodroplets placed on top of individual spots of the microarray by a printing robot. Conversion was followed by lectin binding specific for the terminal sugars. This enzymatic extension of surface-bound ligands in nanodroplets reduces the amount of precious glycosyltransferases needed by seven orders of magnitude relative to reactions carried out in the solution phase. Moreover, only those ligands that have been shown to be substrates to a specific glycosyltransferase can be individually chosen for elongation on the array. The methodology described here, combining focused modular synthesis and nanoscale on-chip enzymatic elongation, could open the way for the much needed rapid construction of large synthetic glycan arrays.
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Glicosiltransferases/metabolismo , Polissacarídeos/síntese química , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Canavalia/química , Sequência de Carboidratos , Glicosilação , Griffonia/química , Humanos , Estrutura Molecular , Nanoestruturas/química , Pichia/genética , Pichia/metabolismo , Lectinas de Plantas/química , Polissacarídeos/química , Aglutininas do Germe de Trigo/químicaRESUMO
The self-assembly of Zn-bisporphyrin tweezers induced by coordination to enantiopure 1,2-diaminocyclohexane features supramolecular chirality induction and inversion processes that are exclusively controlled by the stoichiometry of the assembled complexes.
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Porfirinas/química , Zinco/química , Dicroísmo Circular , Substâncias Macromoleculares/química , Modelos Moleculares , Estrutura Molecular , Análise Espectral , EstereoisomerismoRESUMO
[reaction: see text] The asymmetric aza-Michael reaction of metal benzylamides to alpha,beta-unsaturated amides derived from the chiral amino alcohol (S,S)-(+)-pseudoephedrine has been studied in detail. A deep study of the most important experimental parameters (solvent, temperature, nucleophile structure, influence of additives) has been carried out, showing that the reaction usually proceeds with good yields and diastereoselectivities, although the experimental conditions have to be modified depending on the substitution pattern of the conjugate acceptor. Additionally, a very interesting facial selectivity inversion has been observed when manipulating the structure of the chiral auxiliary, which has allowed a diastereodivergent procedure to be set up for performing asymmetric aza-Michael reactions using the same chirality source. Finally, the adducts obtained in the asymmetric aza-Michael reaction have proven to be very versatile synthetic intermediates in the preparation of other interesting compounds such as beta-amino esters, gamma-amino alcohols, and beta-amino ketones in highly enantioenriched form.
Assuntos
Compostos Aza/química , Efedrina/química , Amidas/química , Aminação , Aminoácidos/síntese química , Aminoácidos/química , Amino Álcoois/química , Benzamidas/química , Ésteres/química , Cetonas/química , Lítio/química , Estrutura Molecular , EstereoisomerismoRESUMO
Reaction of monosubstituted Keggin polyoxometalates (POMs) and [Cu(ac)(pmdien)]+ generated in situ led to the formation of the hybrid metal organic-inorganic compound K5[Cu(ac)(pmdien)][SiW11CuO39].12H2O; its crystal structure and magnetic properties have also been determined. The packing of this compound can be viewed as a stacking of hydrogen-bonded chiral double chains, with the cationic complexes located between the two-dimensional arrangement of POM double chains. DFT calculations performed on [Cu(ac)(pmdien)]+ suggest that the distortion presented in this cationic copper complex is due to electronic effects. An AIM stability study of the cationic copper complex, in order to determine the relationship between the bond angle Cu-O-C and the denticity of the acetate ligand, has been carried out. Topological analyses over the polyhedral distortion, both of the monosubstituted polyanion and copper complexes, have been performed by means of continuous shape measures (CSM).
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Cobre/química , Polímeros/química , Polímeros/síntese química , Compostos de Tungstênio/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Magnetismo , Conformação Molecular , Temperatura , Compostos de Tungstênio/síntese químicaRESUMO
Chiral nonracemic beta-amino esters were prepared in good yields and enantioselectivities using the diastereoselective conjugate addition of nitrogen nucleophiles to alpha,beta-unsaturated amides derived from (S,S)-(+)-pseudoephedrine as the key step. In this way, several beta-amino amide adducts were prepared using different conjugate acceptors and two different lithium benzylamides as nucleophiles. These adducts were easily converted in only one step, into the final, highly enantioenriched beta-amino esters